Biochemistry

Laboratory Manual
BIOLOGICAL SCIENCES M114L
Spring 2015

By

Pavan Kadandle
Julia Massimelli
Brian Sato
Mi Lay
Barbara Woolfolk

University of California, Irvine

Ayala School of Biological Sciences
 COPYRIGHT
Unless otherwise indicated, all materials on these pages are copyrighted by Barbara
Woolfolk and Brian Sato. All rights reserved. No part of these pages, either text or
image may be used for any purpose other than personal use. Therefore, reproduction,
modification, storage in a retrieval system or retransmission, in any form by any means,
electronic, mechanical or otherwise, for reasons other than personal use, is strictly
prohibited without prior written permission.
 Table of contents
SAFETY AND LAB CITIZENSHIP ....................................................................................................... I
HOW TO USE A MICROPIPETTOR ................................................................................................. III
USING THE MICROPIPETTOR ...................................................................................................................... III
ATTACHING THE DISPOSABLE TIP AND TRANSFERRING LIQUID.................................... V
PIPETTING SOLUTIONS USING MICROPIPETTORS..................................................................................... V
WEEK 1 ....................................................................................................................................................... 1
EXPERIMENT 1A – PIPETTING EXERCISE ............................................................................................ 2
INTRODUCTION TO CYTOCHROME P450BM3 ............................................................................................ 3
BACTERIAL PROTEIN EXPRESSION SYSTEMS .......................................................................................... 6
EXPERIMENT 1B – QIAGEN PLASMID PREP .......................................................................................... 9
EXPERIMENT 1C - RESTRICTION DIGESTS .......................................................................................... 13
PREPARATION OF CELL FREE EXTRACTS ................................................................................................ 14
EXPERIMENT 1D - PREPARATION OF CELL FREE EXTRACTS ............................................................ 16
WEEK 2 .................................................................................................................................................... 19
ANALYSIS OF RESTRICTION ENZYME DIGESTS OF PLASMID A AND B ..............................................20
EXPERIMENT 2A – GEL ELECTROPHORESIS .....................................................................................20
ANALYSIS OF CELL FREE EXTRACTS FOR CYTOCHROME P450BM3 ................................................ 22
BEER’S LAW ................................................................................................................................................. 23
SPECTROPHOTOMETER INSTRUCTIONS .................................................................................................. 24
BRADFORD ASSAY ....................................................................................................................................... 25
ABSORPTION SPECTRUM SCAN ................................................................................................................ 26
ENZYME ACTIVITY ASSAY ......................................................................................................................... 26
EXPERIMENT 2B - BRADFORD ASSAY ON CELL FREE EXTRACTS A & B........................................ 27
EXPERIMENT 2C - ABSORPTION SPECTRUM SCAN ON CELL FREE EXTRACTS OF A AND B ........ 31
EXPERIMENT 2D - ENZYME ACTIVITY OF CELL FREE EXTRACTS A & B ...................................... 32
WEEK 3 ....................................................................................................................................................37
EXPERIMENT 3 - AMMONIUM SULFATE SATURATION OF CELL EXTRACT .................................... 40
WEEK 4 ................................................................................................................................................... 46
CHROMATOGRAPHY .................................................................................................................................... 47
EXPERIMENT 4A - GEL FILTRATION CHROMATOGRAPHY ............................................................... 50
POLYACRYLAMIDE GEL ELECTROPHORESIS .......................................................................................... 52
EXPERIMENT 4B - SDS-PAGE ............................................................................................................ 54
WEEK 5.................................................................................................................................................... 58
DETERMINATION OF KM AND VMAX ........................................................................................................... 59
EXPERIMENT 5A - EFFECT OF ENZYME CONCENTRATION ON THE RATE OF THE REACTION .... 63
EXPERIMENT 5B - DETERMINE KM VALUES FOR LAURIC ACID AND MYRISTIC ACID .................. 64
WEEK 6 ................................................................................................................................................... 68
KINETIC PATTERNS OF ENZYME INHIBITION ........................................................................................ 69
EXPERIMENT 6A – DETERMINATION OF [I]50 .................................................................................. 72
EXPERIMENT 6B – INCREASING [S] TO OBSERVE THE EFFECT ON PERCENT INHIBITION ........ 74
EXPERIMENT 6C – DETERMINE THE KI ............................................................................................. 75
WEEK 7.....................................................................................................................................................78
LYSOZYME .................................................................................................................................................... 79
EXPERIMENT 7A – GENERATE EXTRACTS CONTAINING LYSOZYME FROM EGG WHITES .......... 80
EXPERIMENT 7B – BRADFORD ASSAY TO DETERMINE PROTEIN CONCENTRATION..................... 81
EXPERIMENT 7C – PERFORM AN ENZYME ACTIVITY ASSAY WITH M. LUTEUS CULTURES ....... 83
PROTEIN CRYSTALLOGRAPHY................................................................................................................... 84
EXPERIMENT 7D – SET UP CRYSTALLIZATION TRAYS WITH PURIFIED LYSOZYME ..................... 87
WEEK 8 ................................................................................................................................................... 89
MICROSCOPE BASICS ..................................................................................................................................90
USING THE OCULAR MICROMETER IN YOUR MICROSCOPE .................................................................. 91
EXPERIMENT 8A – OBSERVE CRYSTAL FORMATION........................................................................ 92
CRYSTALLIZATION VARIABLES ................................................................................................................. 94
EXPERIMENT 8B – DEVISE NEW CONDITIONS TO CRYSTALLIZE LYSOZYME ............................... 95
WEEK 9 ................................................................................................................................................. 100
EXPERIMENT 9 – OBSERVE CRYSTAL FORMATION AND FILL OUT THE CRYSTALLIZATION
WORKSHEET ...............................................................................................................................................100
Safety and lab citizenship
THINK ABOUT EVERYTHING YOU ARE DOING, and judiciously use your common
sense. Be aware that the lab is fraught with potential hazards, and know that you are just
one stupid accident away from causing yourself or others around you serious injury.
You will be advised when chemicals you are working with are hazardous, either by your
instructor, or as noted in the lab manual. PAY ATTENTION to these warnings, and be
extra careful when working with these reagents. Working in lab is NOT like driving:
 You cannot text.
 You cannot call your BFF.
 You cannot invite your BFF (or anyone else!) to come with you to lab; entrance into the lab is
at the discretion of the TA\Instructor\Lab coordinator.
 You cannot eat, smoke, put on makeup or drink in lab.
 You cannot be drunk or high on any kind of drug in lab. Not even medicinal marijuana. You
can be high on life.
 You MUST wear closed-toed shoes in lab.
 You must wear your lab coat AT ALL TIMES in lab.
 DO NOT mouth pipette in lab.
 Clean your bench and common areas before you leave.
 If you make a mess, clean it up. If you are reported creating a mess and NOT cleaning it up,
after one warning, you will be penalized 10% of your final score. There are other people
working in lab, and other sections using the lab, and it is EVERYONE'S responsibility to
maintain the lab and equipment.
 If you break a piece of equipment (or notice that something is not working), report it
immediately.
 Supplies to bring to lab:
o Lab coat (lab apron not acceptable)
o Wear long pants, close toed shoes
o Lab manual
o Pens
o Scientific calculator/laptop (if you have one, helps with graphing)
 Bacterial cultures are provided in lab, take the time to use safe microbiological techniques:
o Swab benches with disinfectant provided before and after lab sessions
o When pipetting bacterial cultures be sure waste ends up in proper location.
 DO NOT MARK GLASSWARE AND PLASTIC BEAKERS WITH FELT PENS:

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 o Use tape which can then be marked with felt pen, pencil, and/or ball-point pen. For
plastic Sorvall tubes and eppendorf tubes, mark with a felt tip pen as taped tubes will
not fit in the rotor.
 The TA will explain the operation of the Beckman spectrophotometers and you will use the
instruments under their direction. Read all instructions regarding the use of the
spectrophotometer in the lab manual.
 The TA will operate Sorvall super speed centrifuges.
 Safety is of the utmost importance. If you have any questions or notice anything unsafe
please report immediately to your TA, Mi Lay or Dr. K.

Cleaning up
It is mandatory that each student pick-up after himself or herself after each experiment.
This means wiping off benches, disposing of dirty glassware properly, putting away test
tube racks, etc. Please take time to do the following so implementation and clean-up of
lab experiments will be easier on you, your TA, and staff:
 Make sure all of your protein extracts or other reagents necessary for future labs are placed
in the proper location. If this is not followed you may not be able to complete future
experiments.
 Remove all tape from test tubes, flasks, pipettes, and plastic ware after completion of your
experiments. Remove all writing from glass and plastic ware after your experiment.
 Empty contents of test tubes and/or cuvettes into clearly marked waste bottles unless
instructed otherwise. Rinse tubes before placing in the dirty dish cart. Dispose of cuvettes in
the biohazard waste.
 Place plastic beakers (small and large) in the dirty dish cart. Eppendorf tubes should be
disposed of in the biohazard waste at the front of the room.
 Flasks with media/cells still remaining: Do not put contents down the sink. Place flasks with
contents (without tape) in the tub marked "Contaminated Flasks." This tub will be autoclaved
to kill bacterial culture and then glassware will be washed and used again.
 Place disposable glass serological pipettes and Pasteur pipettes in biohazard waste.
 All broken glass should also go in the red container marked "Disposable Glass."
 Tips used with micropipettors should be disposed of in the biohazard waste at the front of the
room.
 Gloves can be disposed of in the regular trash.
 Swab your bench clean, and put away all equipment.
DO NOT dispose of any solutions in the sink without first checking with your
instructor.

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How to use a micropipettor
The Pipetman micropipettors available in M114L lab cover four volume ranges:
0.5 μl – 10 μl (the “P-10”) 2 – 20μl (the "P-20"), 20 – 200μl (the "P-200") and
200 – 1000μl (the "P-1000"). You must choose the correct one for the volume to be
measured. The lowest end of each volume range is subject to the greatest percentage
error. Thus, to pipette 20μl, you would choose the P-20 rather than the P-200.
For accuracy, you must stay within the calibrated range of each micropipettor. The
volume setting is determined by rotation of the calibrated volume adjustment (stroke)
knob near the top of the micropipettor.
Micropipettors make use of disposable plastic tips that
attach to their ends. It is these tips that actually contain
the measured volume of solution.
Please handle these devices carefully. They are
expensive and have many small internal parts, and
since they work with such small volume units
(1μl = 10-6 liters), they must be used correctly. Never
open or remove any part of the micropipetor!
Pipet tips. The tips can be purchased
with or without the filter barrier
Never let the plunger snap up!
(white disk).
http://news.thomasnet.com/images/large/
825/825855.jpg

Using the micropipettor

Test the plunger, which has two distinct stops
when depressed. The first stop is used for
drawing liquid up into the tip, i.e., the plunger
is moved to the first stop and the tip is then
immersed in the solution. The plunger is then
allowed to return to its starting position (by
slowly releasing it).
The second stop (plunger pressed all the way
down) is used to dispense measured volumes
from the pipettor. Hold the end of the tip over
a tube. Move the plunger to the second stop to
empty the liquid, then let it return slowly to its
Parts of the pipet.
starting position. http://www.rainin.com/pdf/pipetman_manual.pdf

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NOTES:
(a) Always use a new pipette tip for each new sample.
(b) It is possible to mix small volumes of liquids by repeated micropipetting. This is
achieved by pressing the plunger up and down several times to mix the solutions.
Examples of volumes set up:
To pipette 2.5 l
Using the P10 or P20, the black hairline must be aligned such that the adjusting wheel
reads black number = 2 and red number = 5

Other examples:

P1000 reading 350 µL P20 reading 2.5 µL P200 reading 95 µL

How to set and read a pipettor’s volume. wwwn.cdc.gov/dls/ILA/cd/zambia/files/Micropipettes.ppt

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Pipetting solutions using
micropipettors
 Adjust the pipettor first. Set the
micropipettor to the appropriate volume.
See the previous section for setting
volumes.
 All the pipet tips have been sterilized.
Therefore it is important not to
contaminate the tips while placing them on
the pipettor. The lid of the pipet box
should be taped on one side so that the lid
opens like a music box. This will prevent
the lid from being lost or contaminated. To
pull a tip from the pipet box, jam the tip
onto the pipettor using a little pressure.
Then lift the pipette up. Do not just pull
towards you as that will bend the end of
the pipette causing the plastic to
eventually snap (this happens all the
time!).
 Place the appropriate amount of pressure
on the pipet plunger.
Attaching the disposable tip and transferring liquid.
o There are two pressure points on http://www.rainin.com/pdf/pipetman_manual.pdf
the pipet plunger. The first
pressure point is used to aliquot the appropriate volume and the second pressure
point is used to completely push the total volume out of the pipet tip.
 Steps to using the micropipettor
o Place only the very end of the pipet tip into the solution and gently release the
pressure on the plunger. If the pressure is released too rapidly, the liquid in the tip
will squirt onto the end of the micropipettor contaminating the liquid in the pipet tip.
o Pull the pipettor out of the solute and place the pipette tip into the solvent and down
fully on the plunger. Release then press again a few times to rinse the tip. Finally,
press the plunger all the way down (to the second pressure point). Keeping the
pressure on the plunger remove the micropipettor from the tube.
o NOTE! If you release the plunger while the tip is in the solution, you will pipet the
solution into the tip again.
o To eject the tip, simply push down on the small white plunger. (If this does not work,
simply pull the tip off)
o
DO NOT USE THE SAME PIPET TIP IN TWO DIFFERENT
SOLUTIONS

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Week 1
A. Pipetting Exercise
B. Plasmid Mini-Prep to Extract Plasmid DNA from Transformed E. coli
C. Restriction Digest of Plasmid DNA
D. Preparation of Cell-Free Extracts from Transformed Escherichia coli

Definitions:
Plasmid:
An autonomous, self-replicating extrachromosomal DNA molecule

Plasmid vector:
A DNA molecule that can carry inserted DNA and be perpetuated in a host cell

Expression plasmid:
A cloning vector that has been constructed in such a way that after insertion of a DNA
molecule, its coding sequence is properly transcribed and the RNA is efficiently
translated. The cloned gene is put under control of a promoter sequence for the
initiation of transcription and often also has a transcription termination sequence at its
end.

palindromic sequences:
Complimentary DNA sequences that are the same when read in each direction (e.g. 5' to
3'). These sequences are recognition sites for type II restriction enzymes.

polylinker:
A synthetic DNA sequence that contains a number of different restriction enzyme sites
(also called multiple cloning site)

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EXPERIMENT 1A – Pipetting Exercise

REAGENTS:
24 well dish
Dye solution
Water

PROCEDURE:
For this exercise, you will dilute a solution containing a dye. There will be 4 dilutions in
total, generated through serial dilutions (making a dilution from a previously diluted
solution). With each dilution, the solution will appear lighter in color. Each person in
the group will perform this once to ensure that they are comfortable doing so.
Each dilution will be a 1:5 dilution with a total volume of 1 ml.
 Fill a small beaker with water from the sink.
 Add 800l water to the first well in your 24 well dish.
 Add 200l of the concentrated dye solution to the water. Before proceeding to the next
dilution, it is important to mix this solution so that it is homogeneous. When mixing with a
pipettor, a general rule of thumb is to set the pipettor at roughly 75% of the total volume of the
solution, which in this case is 750l. Slowly pipette up and down 4-5 times to mix.
 Repeat steps 1 and 2 until you have 4 wells filled water and dye. The solution in each well
should be lighter than the previous.
 If the results are ambiguous, please repeat. It is important that you can accurately pipette for
the exercises this week and the rest of the quarter.

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Introduction to Cytochrome P450BM3
The experiments covering over half of this course will focus on the prokaryotic enzyme
cytochrome P450BM3. P450 proteins are a highly conserved family, expressed in
bacteria all the way up to humans. There are hundreds of different enzymes in the P450
family and while conserved, they play a role in many different functions, including
steroid production, drug breakdown, vitamin production, and cholesterol metabolism,
among others.
P450 proteins are pigmented (P for pigment), and you
will notice that your bacterial extract containing P450 is
a reddish-brown color. This is due to the presence of a
heme prosthetic group containing a central iron atom.
In the heme group of cytochrome P450s, the iron is
bound to four nitrogen atoms and a cysteine, as can be
seen in Figure 1. The heme group is important for the
enzyme function, as we will describe shortly. In its
resting state, the heme group gives a visual absorption Figure 1. Heme group of a
spectrum similar to that of the cytochrome proteins. cytochrome P450 protein.
(From the coordination chemistry page
Technically, P450 proteins are not cytochromes, as of Saint John’s University.)
cytochromes are factors involved in the electron
transport chain. Still, P450s are commonly referred to as cytochrome P450s. The
cytochrome P450 that we are specifically studying is P450BM3, from Bacillus megaterium
(BM). BM3 is one of the best understood P450 proteins in the family.
Despite their different cellular functions, a common P450 enzymatic activity underlies
each. P450 proteins function as hydroxylases (or monooxygenases), which catalyze the
replacement of a substrate’s hydrogen with a hydroxyl group. The hydroxyl is derived
from molecular oxygen. A generic hydroxylase reaction is shown in Figure 2. The term
oxygenase refers to enzymes which introduce oxygen into a substrate directly from
molecular oxygen (and not water). P450s are monoxygenases as opposed to
dioxygenases, as only one of the oxygen atoms is used.

Figure 2. A standard hydroxylation reaction. From Nebert et al. Ann Rev Biochem. 1987.

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Different P450 enzymes interact with
different substrates. The cytochrome
P450BM3 substrate that will be used in
lab is lauric acid, a 12 carbon
saturated fatty acid, the structure of Figure 3. The structure of lauric acid.
which is seen in Figure 3. Modified from www.pherobase.com.

Substrate hydroxylation involves the transfer of electrons. One oxygen atom in the
molecular oxygen (O2) molecule is reduced to a hydroxyl group, while the second is
reduced to water. In order for this to occur, electrons are required, and are passed to
the enzyme’s active site by a variety of electron carriers, primarily NADH (nicotinamide
adenine dinucleotide) or NADPH (nicotinamide adenine dinucleotide phosphate).
NADH/NADPH is oxidized to NAD+/NADP+ through the removal of two electrons. These
electrons are utilized in the hydroxylation reaction. Different P450 enzymes work in
conjunction with different electron carriers. For P450BM3, we will use NADPH.
There are two classes of P450 enzymes. Class I enzymes generally include
mitochondrial and bacterial P450s. FAD (flavin adenine dinucleotide) groups and iron-
sulfur proteins are responsible for electron transfer in this class. Class II enzymes are
primarily eukaryotic and consist mostly of membrane bound P450s that receive
electrons from FAD and FMN (flavin mononucleotide) groups.
P450 enzymes are generally 2 or 3 polypeptide systems that work in conjunction.
Separate polypeptides can encode for the hydroxylase activity (containing the heme
domain), the FAD/FMN domains, and the NADPH binding domain. Many of the class I
enzymes are three protein systems while class II enzymes tend to be 2 protein systems.
Cytochrome P450BM3 is an outlier from these guidelines. BM3 is the only known
bacterial class II P450. It also consists of a single protein, which contains the heme
group, an FAD and FMN. In addition to electron transfer, the FAD domain is also
responsible for NADPH binding. While a single protein, it appears that the BM3
structure consists of two distinct domains. When the protein is cleaved into two pieces,
one with the heme group and the other with the FAD/FMN, substrate hydroxylation can
still occur, albeit at a slower rate. Of interest, P450BM3 enzymatic activity is much more
rapid than most other P450 systems. This may have to do with the fact that all of the
protein components are found in a single polypeptide.
Figure 4 summarizes the model for how P450 proteins are capable of substrate
hydroxylation. The exact mechanism is not yet known, as some of the intermediates
below are very transiently formed and have not been definitively identified. Each step
in the cycle will be described below. Please note that for P450BM3 the substrate (R-H) is
lauric acid.

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The heme group starts in the
ferric iron state with the iron
bound to a water molecule. The
substrate (R-H) associates with
the P450 enzyme, expelling the
water.
FAD or FMN, which received
electrons from NADPH, passes
an electron to the heme domain,
reducing the iron from Fe3+ to
Fe2+.
The iron then binds to an O2
molecule.
The P450 molecule receives
another electron. One of the
oxygen molecules is reduced
resulting in the formation of a
superoxide-ferric iron complex. Figure 4. Model of P450 hydroxylation.
The superoxide is then Modified from Munro et al. Biochimica Biophysics Acta. 2006.

protonated.
The hydroxyl group is protonated producing water, releasing one of the oxygen atoms.
The remaining oxygen attached to the iron group is now a free radical, which allows it to
interact with the substrate. The substrate is then released with an added hydroxyl
group. The cycle will then repeat itself with a new substrate molecule.
In lab, you will be working with P450BM3 that has been synthesized by E. coli as
described below. Each group will be given two tubes of E. coli, one expressing P450 and
one expressing an empty vector (no P450 gene). Once you determine which bacterial
extract contains your P450 protein, you will begin purification of that extract for later
kinetics studies.

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Bacterial Protein Expression Systems
Plasmid and E. coli Background
The first few experiments in lab will focus on two bacterial plasmids, one, which
encodes for our protein of interest, cytochrome P450, and one, which does not.
Plasmids are small circular double stranded pieces of DNA. They are not incorporated
to the main bacterial chromosome and amplification occurs separately from normal
cellular DNA replication. They are also not essential for cell viability.
Plasmids contain a number of components needed for replication and expression.
These components include:
1. Origin of replication
2. Drug resistance marker
3. Polylinker
The origin of replication is the site at which replication of the plasmid is initiated. It is
here that DNA helicase, DNA polymerase, and other factors involved in replication bind.
Transformation, the process of adding foreign DNA (in this case the plasmid) to cells, is
highly inefficient. Roughly 1 in 10,000 bacterial cells will gain the plasmid through the
transformation process. To identify those that have done so, we use a selection process
that inhibits the growth of untransformed cells. In many cases, this selection involves
the addition of antibiotics, such as the drug ampicillin, which inhibits cell wall synthesis
thus preventing bacterial growth. To utilize antibiotics as a selection, an ampicillin
resistance gene has been added to our plasmids. When cells take up the plasmid, they
express a protein which breaks down the antibiotics. Thus, cells with the plasmid grow
in the presence of ampicillin.
Finally, the polylinker is a region added to plasmids that contains a number of unique
restriction sites. This facilitates the cloning process, and makes it easier to insert novel
DNA into the plasmid using restriction enzymes. This will be discussed in further
detail, later in the manual.
At the start of lab you will be provided with two versions of your E. coli, which have
been transformed with a different plasmid, either an empty vector (pET3) or the pET3
vector containing the P450BM3 gene (pT7BM3). The E. coli we are using has unique
characteristics:
1. The lac I gene (lac repressor), the lac promoter (lactose inducible promoter) and the
T7 RNA polymerase gene are all integrated into the bacterial chromosome.
2. The strain is sensitive to ampicillin
The fact that the strain is inherently sensitive to ampicillin is important for selecting
which cells have taken up our plasmid of interest. The presence of a lac repressor, lac
promoter, and T7 RNA polymerase is key for the transcription of foreign genes, like
P450, and will be discussed below.

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The pET3 vector is a plasmid
developed for cloning and
expressing target genes
under control of the T7
promoter (pET is plasmid for
expression by T7 RNA
polymerase). It has a T7
RNA polymerase promoter
(a very high expression promoter); and
a gene for ampicillin resistance.
The plasmid pT7BM3 is identical to the
pET3a vector but inserted downstream
of the T7 promoter is the Cytochrome P-
450 gene.
Bacillus megaterium has a gene that
encodes for cytochrome P450 but E.coli
does not. To generate pT7BM3, the
P450 gene was excised from a Bacillus chromosome and inserted into the pET3 plasmid
behind the T7 promoter. Both the empty vector (pET3) and the P450-containing
plasmids (pT7BM3) were then transformed into E.coli.

Lactose-Inducible Protein Expression System

Do you remember the lac operon? E. coli lac operon is regulated by an intrinsic negative
regulator called lacI. In the absence of lactose (basically, when the lac genes are not
needed) lacI binds to the DNA and keeps the expression of the lac operon genes (lacZ,
LacY, lacA) OFF. When lactose becomes available for the bacteria to use, lacI is removed
from the lac operon DNA-regulatory regions and transcription of the lac genes occurs.
T7 polymerase is not naturally produced by E. coli; the lac promoter-T7polymerase
cassette was inserted in the chromosome of E. coli. Basically, this bacteria has been
engineered so that expression of T7 RNA polymerase is regulated by the lac promoter.
Normally, transcription is repressed at the lac promoter (absence of lactose), as the lac
repressor (lacI) binds to this region, preventing RNA polymerase from interacting with
the promoter. Lac repressor binding to the lac promoter can be alleviated by the
addition of lactose. Lactose molecules bind to the repressor protein causing a
conformational change. The lac repressor can no longer interact with the lac promoter,
and RNA polymerase is free to bind to the promoter, initiating transcription.
Importantly, once the T7 RNA polymerase has been translated, the T7 RNA polymerase
will only transcribe DNA downstream of the T7 promoter. Remember: P450 gene is
under the control of a T7 promoter. The endogenous (normal) E.coli RNA polymerase
does not recognize the T7 promoter. T7 RNA polymerase can transcribe RNA 5 times

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faster than E.coli RNA polymerase, thus large amounts of the enzyme can be produced
in E.coli due to the T7 expression system. T7-dependent transcription is also a regulated
process, so that the protein of interest (P450) is transcribed only when wanted.
Transcription at the lac promoter can also be induced by the addition of isopropyl-beta-
D-thiogalactopyranoside (or IPTG), a lactose analog. This analog is structurally similar
to lactose, but is not recognized by enzymes in the cell that degrade lactose, thus
maintaining high levels of transcription at the lac promoter. Upon addition of IPTG, the
lac promoter is derepressed and allows for expression of T7 RNA polymerase. In turn,
T7 RNA polymerase attaches to the T7 promoter driving P450BM3 mRNA and later
protein synthesis.

Plasmid Mini Prep

Once the plasmid of interest is transformed into E.coli, the bacteria are responsible for
plasmid amplification. These plasmids can be harvested from the bacteria by mini prep.
The following procedure will separate plasmid DNA from the main chromosomal DNA
and other cellular components.
A plasmid is extrachromosomal DNA that may carry genes that give the cell additional
characteristics such as resistance to antibiotics, resistance to heavy metals or
production of toxins. Like chromosomal DNA, plasmids are circular, double stranded
and supercoiled. Plasmid DNA differs from chromosomal DNA in several ways:
1. It is smaller - chromosomal DNA of E.coli has 4,000 kilobases, whereas plasmid DNA
can range in size from a few to several hundred kilobases.
2. While supercoiled, it doesn't nick as easily as chromosomal DNA because of its
smaller size.
The mini prep will involve denaturing the high molecular weight chromosomal DNA
under alkaline conditions. Alkaline lysis method of plasmid isolation was originally
developed by Brinboim and Doly (1979). In this procedure, bacteria containing the
desired plasmid are harvested from culture medium. Suspension of bacteria is made in
isotonic solution. The first buffer contains EDTA to chelate metals. Many proteins
require these metal ions for their activity, for example DNase, an enzyme capable of
hydrolyzing phosphodiester bonds in DNA. EDTA addition protects the plasmid DNA
from degradation by the cellular DNases. The buffer also contains RNAse, an enzyme
which degrades cellular RNA. This ensures that we are only isolating DNA nucleic acid.
The isotonic bacterial suspension obtained by addition of buffer 1 is subsequently
subjected to lysis by an alkaline solution containing a detergent (SDS) and NaOH. This is
done by adding Buffer 2. This combination has been used for more than 20 years for
plasmid isolation. While detergent serves to lyse cells and denature proteins, alkaline
condition denature genomic DNA, plasmid DNA as well as proteins. SDS denatures
proteins which are part of the cell wall and cell membrane, destabilizing these
structures. The cell membrane is now exposed to the hypotonic environment, resulting

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in cell lysis. During lysis, the “cell debris” (broken cell walls, proteins, and denatured
chromosomal DNA) forms large complexes that are coated with SDS. These complexes
fall out of solution (precipitate) when sodium ions are replaced by potassium ions. The
third buffer contains potassium acetate. The high acetate concentration will neutralize
the pH while the high potassium concentration precipitates proteins. Neutralization
results in renaturation of plasmid and genomic DNA. Since plasmid DNA is covalently
closed and small, it reanneals properly and remains in solution in soluble form while
genomic DNA reanneals random, resulting in the formation of precipitate. Precipitate is
separated by high speed centrifugation. Centrifugation removes the vast majority of
chromosomal DNA (it will form a pellet, while plasmid DNA remains soluble). The
plasmid DNA in the supernatant is collected on a silica bead column. The remaining
unwanted components flow through after which the plasmid is eluted for further use.
While you will identify which bacterial culture possesses the P450-containing plasmid
through protein analysis, you will also examine the plasmid DNA itself. By extracting
the DNA from the bacteria with the mini-prep, you can then perform restriction digests
to obtain differing DNA patterns depending on whether the P450 gene is present in the
plasmid.

EXPERIMENT 1B – Qiagen plasmid prep

REAGENTS:
Microcentrifuge tubes
Qiaprep spin column
Buffer P1: resuspension buffer
Buffer P2: lysis buffer
Buffer N3: neutralization buffer
Buffer PB: binding buffer
Buffer PE: wash buffer
Buffer EB: elution buffer

PROCEDURE:
 With a P1000 micropipettor, aliquot 1.5 ml culture A into a microcentrifuge tube. With a fresh
tip, aliquot 1.5 ml culture B into another microcentrifuge tube. Be sure to label tubes with
your initials/group information.
 Using a tabletop microcentrifuge, spin cells for 1 minute at 13,000 rpm. With a 1 ml
micropipettor, discard the supernatant without disturbing the pellet.
 Repeat step 1 twice (step one should be done a total of 3 times) for each culture.

Pg | 9
*Note: all subsequent steps are to be done for both cultures A and B*

 Resuspend the bacterial pellet in 250l Buffer P1 with a micropipettor tip. Make sure that
there are no clumps remaining.
 Add 250l Buffer P2 to the microcentrifuge tube and gently invert 4-6 times to mix, until the
mixture is viscous. Do not allow the lysis reaction to proceed for more than 5 minutes.
 Add 350l Buffer N3 to the microcentrifuge tube and invert the tube immediately (to avoid
localized precipitation) 4-6 times. Mix solution gently but thoroughly. The solution should
become cloudy.
 Centrifuge for 10 minutes at maximum speed. A compact white pellet will form.
 With a micropipettor, carefully apply all the supernatant to a Qiaprep spin column without
disturbing the pellet. Remember to label the removable blue part of the spin columns.
 Centrifuge for 60 seconds. Discard the flow-through.
 Wash the Qiaprep spin column by adding 0.5ml Buffer PB and centrifuging for 60 seconds.
Discard the flow-through.
 Wash Qiaprep spin column by adding 0.75ml Buffer PE and centrifuge for 60 seconds.
 Discard the flow-through and centrifuge for an additional 60 seconds to remove residual
wash buffer. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions.
 Place the Qiaprep column in a clean 1.5ml microcentrifuge tube. To elute the DNA, add 50l
Buffer EB to the column and allow it to stand for 60 seconds.
 Centrifuge for 60 seconds.
 Remove column and save flow-through.
 You will perform a restriction enzyme digest on 5l of the A and B plasmids.


Restriction Enzymes
DNA is made up of a long string of nucleotides connected by phosphodiester bonds.
Restriction endonucleases, enzymes naturally produced by various bacteria, hydrolyze
those phosphodiester bonds. These enzymes recognize a specific sequence of bases in
double stranded DNA and cleave both strands between a particular base pair.
Restriction enzymes recognize a region on the DNA with a palindromic sequence,
meaning the two DNA strands are identical when read in the 5' to 3' direction.
Some enzymes cleave DNA so sticky ends are formed:

And some cleave so blunt ends are produced:

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When you expose plasmid DNA to a restriction enzyme, the enzyme will recognize a
specific DNA sequence and cut it every time it sees this sequence, generating specific
fragments of DNA. Since different DNA molecules contain different patterns of
restriction sites, a plasmid can be characterized by determining the pattern of the
restriction sites, which lie within it. This is called restriction mapping. Using the
restriction enzyme results you and your classmates generate, you will be able to piece
together maps for plasmid A and B.
The enzymes that you will use today and their corresponding recognition sequence are
below:

Restriction Enzyme Recognition site (black arrow indicates cuts)

BamHI 5'. . . G G A T C C . . . 3'

3'. . . C C T A G G . . . 5'

Hindlll 5' . . . A A G C T T. . . 3'

3' . . . T T C G A A . . . 5'

Pstl 5'. . . C T G C A G . . . 3'

3'. . . G A C G T C . . . 5'

Two different DNA strands can be joined together if they are cut with the same
restriction enzyme(s). This is the basis of molecular cloning, and how your P450
plasmid was generated. The gene for cytochrome P450 was cut on one end with EcoRI
and the other end with BamHI. The vector was cut with the same enzymes in the
multiple cloning site downstream from the T7 promoter. The sticky ends made by
EcoRI and BamHI assist in joining the vector and P450 gene, due to the complementary
base pairing that can occur between similar ends. These cohesive ends of the gene and
plasmid are annealed and joined by DNA ligase to generate the P450-containing plasmid
(pT7BM3).

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Once we purify plasmid from each of your bacterial clones, you will use restriction
enzymes to verify the presence/absence of P450 in your plasmids. Often, the size of the
plasmid insert and vector backbone (empty plasmid) are known and thus this technique
can be quickly used to verify your plasmid. Please look at page 51 for the size of your
plasmids. The goal of a diagnostic digest is to cut your plasmid into specific sized pieces
and analyze those resulting fragments by gel electrophoresis. The pattern of the
fragments on the gel can indicate if the plasmid contains the expected size insert
sequence. By selecting the appropriate enzyme(s), one can either linearize the plasmid
to determine the size of the entire construct or excise some or all of the insert from a
plasmid.
Restriction enzyme digests have a number of components, including enzyme, DNA, a
buffer, BSA and RNAse. The buffer is specific for each restriction enzyme, and contains
the correct balance of ions and pH that will allow for optimal enzyme activity. BSA
(bovine serum albumin), which you will use as a standard for the Bradford assay, is
added as some restriction enzymes are sensitive to dilution resulting in an altered the
enzyme structure. Addition of a protein like BSA helps to keep the restriction enzyme in
its folded confirmation. The RNAse is necessary to eliminate any RNA contamination
that may be present in your mini-prep sample.

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Following the completion of the restriction digest, a stop buffer is added to inhibit the
activity of the restriction enzyme. This buffer contains SDS, EDTA, glycerol and
bromophenol blue. Glycerol increases the density of the restriction digest mixture, so
that when it is added to our agarose gel, the samples remain in the well rather than
floating in the water. Bromophenol blue is a dye, which allows us to visualize the
motility of the DNA as it travels through the gel. Based on our use of it in the mini prep,
how do you think SDS and EDTA interfere with enzyme action?
We will run in the gel both uncut and cut plasmid. Note that circular (uncut or
undigested) plasmids are supercoiled. Supercoiled DNA has a more compact and
entangled shape, like a twisted rubber band, than its corresponding non-supercoiled
forms (linear, nicked, see http://bio.classes.ucsc.edu/bio20L/info/animate/anim2/topo.htm).
When supercoiled DNA is cleaved by a restriction enzyme just once it unravels to its
linear form. If supercoiled DNA is nicked (a phosphate bond is broken anywhere in the
molecule in either strand) it completely unravels to form a circle. In your uncut plasmid
sample you will probably see more than one band (mix of supercoiled, linear and nicked
plasmid), check the web page link shown above for clarification.

EXPERIMENT 1C - Restriction digests

REAGENTS:
micropipettes with tips
microcentrifuge tubes
10x restriction buffer
restriction enzymes BamHI, HindIII, PstI
-each group will be assigned 1 restriction enzyme for single digest and 2
restriction enzymes for double digest
plasmid A and B DNA from miniprep
10x STOP buffer: 1% SDS, 15mM EDTA, 50% glycerol, 0.1% bromophenol blue
RNase A (0.1 mg/ml)
sterile water

PROCEDURE:
 Each group will use 5l of their plasmid prep and one of the three restriction enzymes
provided for the single digest and 2 enzymes for the double digest. Four digests total will be
performed (1 single and 1 double with both plasmids A and B).
 The following reagents will already be aliquoted into sterile eppendorf tubes:
o 10l sterile water*
o 2l 10x restriction buffer designated for your particular enzyme
o 5l plasmid DNA (you will add this)
o 1l BSA
o 1l restriction enzyme
o 1l RNase A

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* for a double digest, use 1l of each enzyme will be used along with 9l water
 All of the above ingredients, except plasmid DNA, will be aliquoted into a microcentrifuge
tube before you arrive to lab. Add 5l plasmid DNA into each of the tubes you are assigned.
Label your tubes.
 Place tubes in 37 C water for 1 hour.
 After incubation, add 2l STOP buffer to each tube and mix by flicking the tube. Give them to
your TA to stick in the freezer. Next week you will run the sample on an agarose gel to
separate and visualize the DNA.

Preparation of Cell Free Extracts

In your experiment today, the transformed E.coli cells were grown overnight in Luria
broth and ampicillin. What is the purpose of the ampicillin? IPTG and more ampicillin
were then added and the cells continued growing for 4 hours at 37C.
Your task today will be to make cell free extracts of culture A (has bacteria with plasmid
A) and culture B (has bacteria with plasmid B). You will conduct a number of tests to
determine which bacteria express the P450 plasmid. Enzyme purification and
characterization will continue only with the P450-containing extract.
To make cell free extracts, you need to break apart the E.coli, a Gram negative
bacterium. Gram negative bacteria have several layers to its cell wall. The inner layer is
known as the peptidoglycan layer and gives the cell its shape and rigidity. The outer
layer is known as the lipopolysaccharide layer and acts as a permeability barrier like the
cytoplasmic membrane.
Gram negative cell
envelope. The cell envelope
is defined as the outer layers
surrounding the cytoplasm
of a cell and includes the
cytoplasmic membrane and
cell wall. The cell wall is
defined as the rigid layer
outside the cytoplasmic
membrane. The cell wall of
Gram negative bacteria has
two layers: inner
peptidoglycan and outer
lipopolysaccharide
In order to release the cell
contents, you will need to

Pg | 14
disrupt the cell wall. First, an extraction buffer is added containing EDTA, DTT and
PMSF. EDTA chelates metal ions necessary for the structure of cell wall proteins, thus
weakening the outer layer. This allows lysozyme to access the peptidoglycan layer.
Dithiothreitol (DTT) is a reducing agent that counteracts oxidation of proteins, for
example cysteine residues, preventing protein aggregation. Phenylmethylsulfonyl
fluoride (PMSF) inhibits the action of proteases, which can degrade your protein of
interest during the purification process.
Lysozyme is then added, which hydrolyzes peptidoglycan by breaking the glycosidic
bonds between N-acetylglucosamine and N-acetylmuramic acid, the amino sugars.
Water enters the degraded cell wall and the cell swells and lyses due to the disruption of
the cytoplasmic membrane. Lysozyme will be the subject of crystallography
experiments performed later in the quarter.
Once the cell wall is disrupted, the cell suspension is put through a freeze/thaw cycle to
break any spheroplasts that remain (cells without a cell wall but with a functional
plasma membrane). After centrifugation, the pellet consists of cell wall and cytoplasmic
membrane debris and the supernatant has the cytoplasmic contents including soluble
proteins like P-450BM3.

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EXPERIMENT 1D - Preparation of cell free extracts
REAGENTS:
Extraction buffer: 50 mM potassium phosphate, pH 7.4
10 mM EDTA
1 mM DTT
0.1 mM PMSF
Lysozyme: 10 mg/ml
50ml size Sorvall tube with pellet from culture A (has plasmid A)
50ml size Sorvall tube with pellet from culture B (has plasmid B)
2 clean 50ml size Sorvall tubes
common container with dry ice and alcohol for the freeze step
10ml graduated cylinder
2 conical plastic tubes with lid: one labeled “A” and the other labeled “B”

PROCEDURE:
****************************************************************************************
BE AWARE THAT UNLESS STATED OTHERWISE ALL TUBES CONTAINING THE
ENZYME SHOULD BE KEPT ON ICE. SOME ENZYMES ARE VERY SUSCEPTIBLE TO
DENATURATION IF NOT KEPT COLD. EACH PAIR WILL BE ISSUED A SMALL ICE
BUCKET TO KEEP SAMPLES ON ICE.
****************************************************************************************
 Each group will receive one 50 ml Sorvall tube with a pellet from culture A and one 50 ml
Sorvall tube with a pellet from culture B. Weigh each tube, and write this value in the table
below.
 Weigh 2 fresh empty Sorvall tubes. Write this value in the table below. Subtract this weight
from the weight of the tube with the pellet to obtain the pellet’s weight.
 Fill out the remainder of the table below. Check your values with your TA before proceeding.

Weight of Weight of tube Difference in Total volume ml of lysozyme

empty tube plus pellet weight of cells plus added (1/5 of
(grams) (grams) (grams) extract buffer total volume)

Culture A

Culture B

Pg | 16
 Add a volume of extraction buffer 3 times that of the cell weight (assume 1 ml is equivalent to
1 gram). Resuspend the cells with a wooden stick.
 Add 1/5 volume of lysozyme solution. For example, if your total volume of cell suspension
(cells and extraction buffer) is 6 ml, add 1.2 ml lysozyme. Cap the tube with Parafilm and
make a small opening at the top so that a glass rod is able to fit through it.
 Using the glass rod, mix the tubes continuously while they are on ice. Be sure to mix
vigorously! (do not just stir) This means that over the entire 30 minutes, you are agitating
the extract with the glass rod. Failure to adequately lyse the cells will mean your protein
concentration will be too low, making it difficult to perform the experiments over the next
few weeks. Mixing should resemble beating an egg rather than stirring soup.
 After mixing, immerse each Sorvall tube into a dry ice/alcohol bath. Be careful to avoid
putting your fingers into the freezing mix! Let the tubes sit for 15 minutes so they completely
freeze.
 Thaw the tube in a room temperature water bath. Be careful that the Parafilm on top is
sealed so no water gets into the tube.
 After thawing, centrifuge the tubes for 30 minutes at 20,000 rpm.
 Save the supernatant fraction. Decant the supernatant into a plastic graduated cylinder and
record the volume. Then, pour the liquid into large conical plastic tube with cap. Label each
conical tube. Note if you see a difference in color between the 2 extracts. Keep each tube on
ice as most enzymes denature if not kept cold.

SAVE CELL FREE EXTRACTS A AND B FOR SUBSEQUENT LABS!!

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Practice questions (not for the lab report):
 Describe why the E. coli strain we used is suited for expression of cytochrome P450.
 Can you already tell whether bacteria A or B contains the P450? Explain.
 Consider the hypothetical plasmid pX. To map pX, it is first cut with each of several
restriction enzymes expected to cut relatively infrequently, such as EcoRI, BamHI or EcoRV.
The number and sizes of the resulting fragments are determined by agarose gel analysis
which you will do next week. Assume that the sizes of the resulting fragments are those
given below:
Enzyme used to cut pX Size of resulting fragment(s)
EcoRI 5kb, 6kb
BamHI 3.5 kb, 7.5 kb
 From this, you can deduce that EcoRI cuts pX twice and BamHI cuts it twice. You can also
conclude that the size of the plasmid is 11 kilobases by adding the fragment sizes from a
single digest.
 The results of the next step, cutting pX with combinations of two enzymes to determine the
relative positions of the sites, follows:
Enzymes used to cut pX Sizes of resulting fragments
EcoRI + BamHI 1.5 kb, 2 kb, 3.5 kb, 4 kb
 Draw a restriction map based on the information above.

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WEEK 2
A. Gel Electrophoresis to Analyze Restriction Digest of Plasmids
B. Bradford Assay to Determine Protein Concentration of Bacterial Extracts
C. Absorption Spectrum Scan to Detect the Presence of a Heme Group in Bacterial
Extracts
D. Enzyme Activity Assay to Detect P450 Activity in Bacterial Extracts

Definitions
DNA gel electrophoresis:
Using an agarose gel to separate DNA fragments based on size, the fragments are
mobilized by taking advantage of the inherent negative charge of the DNA helix

absorption spectrum:
Pattern of energy absorption by a substance when light of varying wavelengths passes
through it

prosthetic group:
A metal ion or an organic compound (other than an amino acid) that is covalently bound
to a protein and is essential to its activity

Pg | 19
Analysis of Restriction Enzyme Digests of Plasmid A and B

Last week you digested plasmid A and B with restriction enzymes. This week, you will
see the results of the digest on an agarose gel. Using this data, you will piece together
the maps for plasmid A and B.
Agarose is a polysaccharide that solidifies a solution like jell-o. DNA is placed into the
gel and is subjected to an electrical current (known as electrophoresis). Agarose gel
electrophoresis is based on the fact that DNA is negatively charged at neutral to alkaline
pH due to its phosphate backbone. When placed in an electrical field, in a alkaline buffer
(pH 8), DNA migrates towards the positive pole. The rate at which the DNA moves
towards the positive pole is determined by the pore size of the agarose gel. Agarose
forms a porous lattice and the double-stranded DNA slips through the holes in the lattice
as it moves towards the positive pole. This lattice slows the larger molecules down more
than the smaller ones. The result is the separation of a mixture of DNA fragment by size.
The rate of migration will also depend on the shape of the DNA (supercoiled vs linear vs
relaxed circular). Ethidium bromide is a convenient stain for visualizing DNA in gels. It
is a planar molecule which can insert between the bases in DNA, and when irradiated
with ultra-violet light, the ethidium bromide will fluoresce. The DNA/ethidium bromide
complex absorbs UV at 300nm and reemits visible light in the orange range at 590 nm.
Ethidium bromide is a mutagen so wear disposable gloves when handling it. Two
molecular weight markers will be run on the gels so you can compare the size of the
fragments generated by your restriction enzyme digests to known sized DNA fragments.
The 10x stop buffer was blue due to bromophenol blue which is added as a tracking dye.
Bromophenol blue is equivalent to 100 base pairs in size so it is an indicator of how far a
gel has run.

EXPERIMENT 2A – Gel Electrophoresis

REAGENTS:
Agarose gel tanks
1% agarose gel made up in 1X electrophoresis buffer, ethidium bromide added
20x electrophoresis buffer: 800mM Tris, 200mM NaOAc, 40mM EDTA pH 7.8
Restriction enzyme digests

Pg | 20
PROCEDURE
 Load approximately 20 µL of your sample into the assigned well in the gel.
o With your non-pipetting hand, hold the base of the pipettor and guide the tip over the
well. Try not to insert the tip into the well as you are liable to puncture the bottom of
the gel. Due to the glycerol in the stop buffer, the DNA sample will sink to the bottom
of the well.
o Slowly release the contents of the pipet tip. Hold the plunger down as you remove the
pipettor, or the contents will be sucked back into the tip. Also, try not to expel bubbles
from the pipette tip as this may push some of your sample out of the well.
 The TA will load the two DNA markers: Perfect DNA Markers 0.1-12 kb (5l) and Perfect DNA
Markers 0.05-10 kb (10l).
 As soon as the last sample has been loaded, place the cover on the gel apparatus and plug in
the leads. Remember: red = positive electrode and black = negative electrode. If you mix the
poles up, the DNA will migrate to the top of the gel and out into the buffer.
 Set the power supply at a constant voltage of 60. Any higher and enough heat will build up to
possibly melt the gel.
 Run the gel halfway to two-thirds of the way down. This will allow you to see the smaller
DNA fragments.
 Determine the size of the DNA fragments by comparing them to each marker DNA fragment
in the Perfect DNA Markers 0.1-12 kb and Perfect DNA Markers 0.05-10 kb.

After the gel has been run, your TA will print a picture of it and email the results to the
class. Locate the lanes that show only one band, this is suggestive of this enzyme only
cutting the plasmid once. A single cut will also tell you the approximate size of the
plasmid.
Look at the uncut plasmid, do you see one band or more than one band? Sometimes in
the preparation of plasmids, different forms of the plasmid are obtained, including
nicked, supercoiled, and/or linear. These all migrate at a different distance due to the
shape of the molecule. Supercoiled DNA molecules are more compact and migrate more
rapidly in the gel than the corresponding relaxed molecules.
Look at the lanes that show more than one cut, determine the size of the bands and
make a table. Does the sum of the bands roughly add up to the size of the DNA fragment
which was only cut once?

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DUE WEEK 4:
 A table with the fragment sizes produced by each of the restriction
enzymes used. To calculate fragments sizes, plot a marker standard
curve (see pages 68-69 for instructions).
 Restriction maps of plasmid A and B with the approximate location of
the enzyme cut sites. This map needs to include the relative positions of
the restriction enzyme sites but does not need to include the distances
between each site.
 The grade earned for this assignment will be 3 points of your Lab Report
1 score.

FOR THE LAB REPORT:

When you write your report, you may choose to include the
restriction digest information. For any information regarding the
digests you discuss in the results, be sure to reference the
corresponding data.
Determine the exact size of the DNA fragment by using the two
different DNA ladders to obtain standard migration plots, and use
this to determine the sizes of your DNA fragments. For more detailed
instructions on how to accomplish this, use the resources present on
the internet!

Analysis of Cell Free Extracts for Cytochrome P450BM3

Cytochrome P-450BM3 is a 119 kD protein. It contains one FAD, one FMN, and one heme
group. When the FAD is reduced by NADPH, the FAD passes an electron onto the FMN
and the FMN gives an electron to the heme. Two such electron transfer reactions are
required to complete the hydroxylation reaction. The heme group is capable of binding
substrate and O2. The heme domain generates a characteristic 418nm absorption band
upon reduction in the presence of CO.
Last week you separated the cell envelope of E.coli from its cytoplasmic contents.
Which fraction did you throw away and which fraction did you save? You made 2 cell
free extracts: one from E.coli transformed with the empty vector plasmid (pET3) and the
other from E.coli transformed with the P450-containing plasmid (pT7BM3). Because of
this, only one extract will possess cytochrome P450BM3 activity. Through the
experiments you will perform this week, you will be able to identify which extract (A or
B) contains the enzyme.

Pg | 22
You will be assaying every fraction collected in three ways using a spectrophotometer.
1. Measure protein content via Bradford assay.
2. Do a scan of the absorption of the fractions from 250 nanometers (nm) to 600 nm. The heme
group of cytochrome P-450BM3 absorbs at 418 nm.
3. Assay for enzyme activity by measuring the decrease in absorption at 340 nm, the
wavelength at which NADPH absorbs light. Cytochrome P450BM3 catalyzes the following
reaction:

Beer’s Law
The use of the spectrophotometer for all analyses requires a discussion of Beer's Law
and the extinction coefficient. Various types of photometeric analyses are used in
biochemistry. Colorimeters and spectrophotometers measure the amount of light
absorbed by suspensions while fluorimeters measure the fluorescence produced by
absorbed light. Photometric analyses allow an accurate, sensitive, qualitative, and
quantitative determination of the components of solutions and suspensions.
When white light is passed through a solution containing a compound, the compound
may absorb certain specific wavelengths of light. For example, chlorophyll absorbs blue
and red light while allowing green light to pass through. Thus, solutions of chlorophyll
appear green. Nucleic acid solutions absorb strongly in the ultraviolet region of the
spectrum.
An equation which describes how the intensity of a beam of light diminishes as it moves
through a dissolved substance is the Beer-Lambert Law. This law can be expressed as:

I = Io 10-Ecl
Where “I” is the intensity of light transmitted by the solution, “Io” is the initial intensity
of the beam entering the solution, “E” is the molar extinction coefficient; “c” is the
concentration of the absorbing substrate in moles per liter (M) and “l” is the length of
the optical path in solution centimeters. As it is expressed above, the law generates the
following graph:

Pg | 23
 This graph shows the relationship
between I and Io. Using
differential equations, the
Intensity Solution
following can be derived:
of Concentration
Light moles/liter 𝐈𝟎
𝐋𝐨𝐠 [ ] = 𝐄𝐜𝐥
𝐈
Distance through the solution. 𝐈
The term ⌊ 𝟎 ⌋ is readily measured
𝐈
by means of a spectrophotometer and is usually called the absorbance (Abs) or the
optical density, O.D. Therefore, the above equation may be written O.D. = Ecl.
Absorbance is the log of a ratio and indicates how many orders of magnitude the
transmitted light intensity (I) is less than the initial intensity (Io). For example, an
absorbance of 1.0 means they differ by an order of magnitude. This means the
transmitted light is one-tenth of its original intensity. An absorbance of 2.0 means the
transmitted light is one-hundredth the initial light. Absorbance is unit-less. An
extinction coefficient has units of per mole per liter per centimeter or M-1cm-1.

Spectrophotometer Instructions
The following instructions will assist you with using the spectrophotometer, one of the
main tools in this lab. Spectrophotometers work by shooting light of a specific
wavelength at a sample sitting in a cuvette. The spectrophotometer then assays how
much light is absorbed by the sample, which is displayed as an O.D.
When using the spectrophotometer, you will first normalize the absorbance values. This
is referred to as “blanking” the spectrophotometer. By blanking the spectrophotometer,
your absorbance measurement will be based solely on the molecule of interest and not
due to the absorption caused by any other molecules in solution.
There are two types of spectrophotometer in the lab, DU640 and DU800. Both can be
used for all experiments. Although their operation is similar, there are differences
detailed below.
The following are general instructions for using the spectrophotometer. We will then
detail specific instructions for each type of experiment you will perform with your P450
extract.

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DU640
 Depending on the type of experiment you are performing, pick one of the following options
from the main menu.
 Bradford when performing the Bradford assay
 114L340nm when performing the enzyme activity assay
 114Scan when performing the absorption spectrum scan
 Once in the correct program, insert your cuvette containing the “blank” solution. This cuvette
will be placed in the carrier, in the slot farthest from you.
 At the bottom left of the screen, click blank.

DU800
 The DU800 does not have a main menu. You are able to select different programs and record
sample values on the same screen.
 To choose your program, use the drop down menu at the top left of the screen.
 Fixed Wavelength and then Bradford on the drop down menu when performing the Bradford
assay
 Kinetics and then 1 cuvette on the drop down menu when performing the enzyme activity
assay
 Wavelength Scan when performing the absorption spectrum scan
 Beneath this menu, there is a second drop down menu that will allow you to determine the
specific wavelength used (the Bradford program automatically assays for 595nm light
absorption) or the number of cuvettes needed to scan.
 Once in the correct program, insert your cuvette with your “blank” solution. This cuvette will
be placed in the carrier, in the slot farthest from you.
 At the top middle of the screen, click BLK (blank).

Cuvettes
There are two types of cuvettes you will use, plastic cuvettes (Bradford Assay, Enzyme
Activity Assay) and quartz cuvettes (Absorption Spectrum Scan). When using either,
make sure that they are oriented so that the light from the spectrophotometer passes
completely through the solution. For the plastic cuvettes, there is an arrow on one side,
which faces the light source.

Bradford Assay
 Once you have finished blanking the spectrophotometer, you can now add the cuvettes
holding the first 5 BSA standards. Make sure they are in the correct orientation, with your
first sample in cell 1 in the carrier. The carrier should be placed in the holder so that cell 1 is
farthest away from you.

Pg | 25
  Close the lid of the spectrophotometer. Click the read samples button (for DU640) at the top
left corner of the computer screen. For the DU800, click Go/Read at the top middle of the
screen.
 Once the spectrophotometer is finished reading, remove the cuvettes from the carrier. Repeat
with your next set of samples.
 After you are finished reading your samples, you’ll need to print your results. Click the print
button at the top right corner of the monitor (for DU640). For the DU800, click the file drop
down menu, then print. Please print only one copy per group.
 Clear your data by clicking save/clear. You do not want to save your results, so make sure the
save box is unchecked when the prompt is given (for the DU640). For the DU800, click clear.

Absorption Spectrum Scan

The absorption spectrum scan uses a quartz cuvette instead of the standard plastic
cuvettes. These quartz cuvettes are very expensive, so please handle carefully! This is
necessary because UV rays are incapable of penetrating plastic.

Add 300l of your solution to the quartz cuvette. Use the P200 with gel loading tips. You
will need to pipette twice to load the full 300l. The thin gel loading tips are necessary to
reach the bottom of the quartz cuvette. As you are loading the cuvette, be careful not to
introduce bubbles into the sample. If bubbles are present, gently tap the cuvette to try to
remove them or remove them with your gel loading tip.
Place the cuvette into the carrier and click read samples (for DU640). For the DU800,
click Go/Read at the top middle of the screen.
Once complete, print your results by clicking the print button at the top right corner of
the monitor (for DU640). For the DU800, click the file dropdown menu, then print.
Please print only one copy per group.
Clear your data by clicking save/clear. You do not want to save your results, so make
sure the save box is unchecked when the prompt is given (for the DU640). For the
DU800, click clear.

Enzyme Activity Assay

 Before blanking the DU640, set up the sampling device and number of samples values. For
example, if you want to read 1 cuvette at a time, these numbers should be 1. If you want to
read two, it should be 2, etc. Also, if the screen shows a graph, hit tabulate, which will instead
show you the numerical values for absorbance. For the DU800, this is determined with the
second drop down menu (below the menu that allowed you to choose the program).

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  Once you have blanked the spectrophotometer, keep the lid open. To save time, click the read
samples button (for DU640), which will open the start window. Do not click start yet. This
added step is not necessary for the DU800.
 Tilt your cuvette so it is at a slight angle, and add the NADPH to the side of the cuvette. Do not
add the NADPH directly to the solution. Remember, the assay will begin once the NADPH,
enzyme, and substrate mix, so you do not want that to occur until the last second.
 Cover the top of the cuvette with a small piece of parafilm, and quickly invert the cuvette
twice to mix the reagents.
 Immediately place the cuvette into the carrier, close the lid of the spectrophotometer, and
click start (for DU640) or Go/Read (for DU800). The reaction will carry on for two minutes,
and absorbance values will be recorded every 10 seconds.
 Once complete, print your results by clicking the print button at the top right corner of the
monitor (for DU640). For the DU800, click the file dropdown menu, then print. Please print
only one copy per group.
 Clear your data by clicking save/clear. You do not want to save your results so make sure the
save box is unchecked when the prompt is given (for the DU640). For the DU800, click clear.

EXPERIMENT 2B - Bradford assay on cell free extracts

A&B
Via the Bradford assay, you will analyze the protein concentration of your cell free
extracts. The method measures the absorbance of light by an acidic dye. The dye,
Coomassie Brilliant Blue G-250, has an absorbance peak at 465nm, but when bound to
protein this absorbance shifts to 595nm. The Coomassie dye binds primarily to basic
and aromatic residues (ex. arginine, lysine, tryptophan), so while a general measure of
protein concentration, it can be biased for samples that contain proteins with many or
few basic or aromatic amino acids. Because O.D. is a unitless value, the absorbance
values of your cell free extracts exposed to the Bradford reagent will be compared to a
standard protein curve.
You will use known concentrations of bovine serum albumin (BSA) to set up the
standard curve (see graph below). At the same time you set up a standard curve using
BSA, you will also assay your enzyme fractions for protein content. Realize that each
fraction will contain other proteins in addition to P450 and none of the fractions will
contain BSA which we use as the standard protein. Because of this, it is not an exact
measure of protein levels in the extracts.

Pg | 27
 Sample Bradford Standards Graph

BSA Concentration vs. Absorbance

Absorbance in O.D.
0.6

0.5

0.4

0.3

0.2

0.1

0
0 0.05 0.1 0.15 0.2

Micrograms of BSA in Solution

REAGENTS
Protein Assay reagent (Bradford reagent)
BSA (bovine serum albumin) - 0.5 mg/ml in water
distilled water
plastic cuvettes
Cell free Extract A and B

Acquire a tube of BSA (0.5 mg/ml). For the protein fraction, you will have a final volume
of 1.0ml, although since the amount of BSA added is very small, the volume will not be to
the exact microliter. Make your dilutions with distilled water. Fill in the protocol charts
below. One chart is for the standard curve and the other is for your protein samples.

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STANDARD CURVE USING BSA
Cuvette g BSA l BSA ml H2O ml Bradford Absorbance
reagent

Blank 0 0 0.8 0.2

#1 0.5 1 0.8 0.2

#2 1 0.8 0.2

#3 2 0.8 0.2

#4 4 0.8 0.2

#5 8 0.8 0.2

Because they are more concentrated than the BSA standards, your cell free extracts will
need to be diluted 1:25 in water. If you want to make 50l of the dilution for each extract,
how much water and how much extract will you add?
Show calculations:

CELL FREE EXTRACTS

Cuvette l cell free extract ml H2O ml Bradford Absorbance

(1:25 dilution)
reagent

# 6 Cell free 10 0.8 0.2

extract A

# 7 Cell free 5 0.8 0.2

extract B

* You may have to alter the dilutions depending on the absorbance (OD) of your
extract with the Bradford reagent. If the absorbance is above or below that of your BSA
curve, how will you adjust your dilution? If necessary, see your TA for assistance.

Pg | 29
PROCEDURE:
 Label 7 plastic cuvettes and aliquot the water and protein into each (water first).
 Add the Bradford reagent to each tube, immediately mixing the sample by adding a piece of
Parafilm to seal the top and inverting a few times. It is important that each protein sample is
incubated with the Bradford reagent for the same amount of time. If one sample is incubated
for longer, the Coomassie dye will have more time to interact with the amino acids, which
will result in an artifically high absorbance.
 Wait at least 5 minutes (can be longer).
 Follow the directions for using the spectrophotometer to read absorbance. (below)

Spectrophotometer Instructions
 Click Bradford on the main menu (DU640) or Fixed Wavelength and then Bradford on the
drop down menu (DU800).
 Insert your cuvette with your “blank” solution. This cuvette will be placed in the carrier, in
the slot farthest from you.
 At the bottom left of the screen, click blank (DU640) or BLK (DU800).
 You can now add the first 5 cuvettes holding the first 6 samples in your Bradford assay. Make
sure they are in the correct orientation, with your first sample in the cell 1 in the carrier. The
carrier should be placed in the holder so that cell 1 is farthest away from you.
 Close the lid of the spectrophotometer. Click the read samples (DU640) or Go/Read (DU800)
button.
 Once the spectrophotometer is finished reading, remove the cuvettes from the carrier. Repeat
with your next set of samples.
 After you are finished reading your samples, you’ll need to print your results. Click the print
button (DU640) or file then print (DU800). Please print only one copy per group.
 Clear your data by clicking clear. You do not want to save your results.

When complete, graph the standard curve with the micrograms of BSA on the x-axis and
the absorbance on the y-axis. Hopefully you will have a straight line. Determine the
equation of the best-fit line and plug the absorbance value into the equation. For a
rougher estimate, you can also find the corresponding point on your standard curve and
drop a line down to the x-axis.
Remember the sample dilution you performed. How will this dilution figure into your
calculation of protein concentration? Remember that the micrograms of BSA that you
arrive to based on your standard curve corresponds to the micrograms of protein in the
cuvette, not the original sample.

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EXPERIMENT 2C - Absorption spectrum scan on cell free
extracts of A and B
REAGENTS:
2 micro-quartz cuvettes (careful!)
Cell free extracts A and B
Gel loading tips
PROCEDURE:
 The spectrophotometer should be blanked with extraction buffer first. Use gel loading tips
and your P200 to load the cuvette.
 Add the extracts to the cuvette. Using gel loading tips, aliquot 300l of extract A into the
cuvette located in the 1st compartment of the cuvette holder. Add 300 l of extract B into the
cuvette in the 2nd compartment. Place the holder in the spectrophotometer.
 Consult the directions for using the spectrophotometer. (below)
 When complete, return the extract to your original tube (do not discard it).
 Carefully rinse the cuvettes with water for the next group.

Spectrophotometer Instructions
Click 114LScan on the main menu (DU640) or Wavelength Scan (DU800).
The absorption spectrum scan uses a quartz cuvette instead of the standard plastic
cuvettes. These quartz cuvettes are very expensive, so please handle carefully! This is
necessary because UV rays are incapable of penetrating plastic.

Add 300l extraction buffer to the cuvette as your blank. Use your P200 with gel loading
tips. The thin gel loading tips are needed to reach the bottom of the quartz cuvette. As
you are loading the cuvette, be careful not to introduce bubbles. If bubbles are present,
gently tap the cuvette to try to remove them or remove them with your gel loading tip.
Click blank (DU640) or BLK (DU800).

Add 300l of your solution to the quartz cuvette. Place the cuvette into the carrier and
click read samples (DU640) or Go/Read (DU800).
Once complete, print your results by clicking print (DU640) or file then print (DU800).
Please print only one copy per group.
Clear your data by clicking clear. You do not want to save your results.

Pg | 31
Looking at the scan, where do you see absorption peaks? The heme group should show
a peak at 418 nm. You will also see very high absorption in the ultraviolet region from
260 to 280 nm. The 260 nm peak is due to RNA and DNA. The 280 nm peak is due to
protein.
Do both extract A and B show the same absorption scan? Can you guess which cells
were transformed with the P450 plasmid?

EXPERIMENT 2D - Enzyme activity of cell free extracts

A&B
There are many ways to assay the reaction catalyzed by an enzyme. The most
commonly used methods usually rely on determining the increase in product formed or
the decrease in substrate. One way to follow the activity of the cytochrome P450BM3 is to
follow the oxidation of NADPH. When NADPH is oxidized to NADP+, its absorbance at
340 nm drops. Since there is one molecule of NADPH oxidized for every substrate
molecule hydroxylated, this drop in absorbance at 340 nm is directly proportional to the
rate of substrate hydroxylation. What is the substrate in this enzyme assay? The
extinction coefficient for NADPH at 340 nm is 6220M-1cm-1.
REAGENTS:
100mM potassium phosphate, pH 7.4
10mM lauric acid in 50mM potassium carbonate
10mM NADPH
Cell free extracts A and B

Pg | 32
PROCEDURE:
 The final concentration in the reaction mixture of lauric acid and NADPH is 0.1mM for each.
The total volume of the reaction mixture will be 1.0ml. Using the formula C 1V1 = C2V2,
calculate the volume of each solution needed for the reaction mixture and write it in the
protocol chart below. Check your calculations with your TA.

CELL FREE l LAURIC l NADPH l PO4 TOTAL

EXTRACT ACID PROTEIN BUFFER VOLUME
FRACTION (ml) (ml)

A blank - - - 1.0 1.0

A 10 1.0

B blank - - - 1.0 1.0

B 3 1.0
Depending on the concentrations of your fractions you may have dilute them and adjust the
amount added to the assay. Perform the assay and If the NADPH levels drop too quickly or
slowly, consult your TA about how to adjust the assay contents.

 Aliquot the lauric acid, phosphate buffer, and enzyme into the plastic cuvettes. Do not add
NADPH yet! Place parafilm on top of the cuvette. Incubate at room temperature (not on ice)
for about 2 minutes. Why do you think it is important the reaction is run at room
temperature?
 Read the instructions for using the spectrophotometer below. When you are ready to use the
spectrophotometer, set the number of samples to “1” (for DU640 only).
 Blank the spectrophotometer with the cuvettes lacking NADPH.
 Aliquot _____l NADPH to the enzyme in the cuvette, cover with parafilm, mix quickly,
remove the parafilm, and place the cuvette in the furthest compartment from you. Click start
(DU640) or Go/Read (DU800) on the spectrophotometer. The absorption in the cuvette will be
read every 10 seconds for 2 minutes. Print out the results.
 Repeat for the next cell free extract.

Spectrophotometer Instructions
 Click 114L340nm on the main menu (DU640) or Kinetics and then 1 cuvette on the drop
down menu (DU800).
 Insert your cuvette with your “blank” solution and click blank (DU640) or BLK (DU800).

Pg | 33
  Set up the sampling device and number of samples values to 1 (for this experiment, DU640
only). For the DU800, click 1 cuvette in the second drop down menu. If the screen on the
DU640 shows a graph, hit tabulate, which will instead show you the numerical values for
absorbance.
 Once you have blanked the spectrophotometer, keep the lid open. To save time, click the read
samples button (for DU640) which will cause the start window to open. Do not click start yet.
This is not necessary for the DU800.
 Tilt your cuvette so it is at a slight angle, and add the NADPH to the side of the cuvette. Do not
add the NADPH directly to the solution. Remember, the assay will begin once the NADPH,
enzyme, and substrate mix, so you do not want that to occur until the last second.
 Cover the top of the cuvette with a small piece of Parafilm, and quickly invert the cuvette
twice to mix the reagents.
 Immediately place the cuvette into the carrier, close the lid of the spectrophotometer, and
click start (for DU640) or Go/Read (for DU800). The reaction will carry on for two minutes,
and absorbance values will be recorded every 10 seconds.
 Once complete, print your results by clicking the print button at the top right corner of the
monitor (for DU640). For the DU800, click the file dropdown menu, then print. Please print
only one copy per group.
 Clear your data by clicking save/clear. You do not want to save your results so make sure the
save box is unchecked when the prompt is given (for the DU640). For the DU800, click clear.
Also, be sure to not save the data.

To calculate the rate of NADPH oxidation, make a graph of O.D. versus time. Determine
the best-fit line. The slope of this line will be the rate of the reaction in terms of
O.D./second. To calculate the rate in terms of moles of NADPH oxidized/second, use the
molar extinction coefficient of 6220 M-1cm-1 at 340 nm for NADPH. Remember Beer’s
law, O.D. = Ecl.

l = length of the path light travels through cuvette (1 cm)

E = molar extinction coefficient
c = concentration in molarity
Multiply concentration by reaction volume in liters (1.0 ml = ? liters) to get moles of
NADPH oxidized. Convert to moles of NADPH (1 mole = 10-6 moles).
The purification of a protein is expressed in specific activity. Specific activity should be
expressed in moles of NADPH oxidized/second/mg protein. For this, you will need to
determine how many milligrams of protein were used in this assay.
Total activity is calculated by multiplying total protein in the fraction by specific activity.

SAVE CELL FREE EXTRACTS A AND B FOR SUBSEQUENT LABS.

Pg | 34
Due Week 3 lab:
Fill in Table 1 on the following page.
You will need to include (1) the BSA standard graph and best fit line, (2) all calculations
for extract B (only one set of calculations is necessary as we assume extract A is done
identically), and (3) the enzyme activity graph for extract A and B.
Your TAs will grade your work, and return it to you week 4 along with any necessary
corrections. The week 3 and week 4 data will be analyzed similarly. Because the TAs will
be reviewing this data, calculations for Table 1 and 2 are not necessary to include in lab
report 1. The grade earned for this assignment will be 10 points of your Lab Report 1
score.
FOR THE LAB REPORT:
Fill out and include Table 1 (or a version of it).
Include a figure showing your absorption spectrum scan.

 Practice Problems (Not for lab report)

 You are performing a Bradford Assay. You dilute your protein of interest 1:100 and add 3l of
the dilution to a 1ml Bradford reaction. Based on a BSA standard curve, your O.D. for the
protein of interest is equivalent to 1g protein in the Bradford reaction. What is the
concentration of the original solution containing your protein of interest? Include units.
 For the enzyme activity assay, list each reagent used. What is the purpose of each of these
reagents?

Pg | 35
Table I: Cell Free Extract Characterization Chart

Cell Free Extract A Cell Free Extract B

I
Total extract volume
(ml)

II
Volume of extract used in Bradford
assay (l)

III
O.D. from Bradford assay

IV
mg protein used in Bradford assay
(based on BSA standard curve)

V
Extract concentration
IV/(II x 10-3) (mg/ml)

VI
Total mg protein
IxV

VII
Volume of extract used in enzyme
assay (l)

VIII
 O.D. per second
(slope of best fit line)

IX
moles NADPH oxidized per sec.

X
mg protein used in enzyme assay
(VII x 10-3x V)

XI
Specific Activity
moles NADPH oxidized per sec per
mg protein (IX / X)

XII
Total Activity
Specific activity x Total Protein

(XI x VI)

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WEEK 3
Further Purification of the Cytochrome P450 Extract Through Ammonium Sulfate
Saturation

Definitions

ammonium sulfate fractionation:

Used in the isolation of proteins where the addition of ammonium sulfate to a protein
solution decreases solubility of specific proteins

dialysis:
Removal of small molecules from a solution by allowing them to diffuse through a semi-
permeable membrane into an aqueous solution

Pg | 37
This week you will be using ammonium sulfate addition as a first step in the purification
of a Cytochrome P450BM3 from your crude cell extract. This is an example of the
selective “salting out” of a protein from a solution. The solubility of a protein depends in
large part upon the interaction of the various charged groups at its surface with the
solvent molecules. Generally, the protein exhibits a minimum solubility in the
isoelectric pH region where there is no net positive or negative charge on the molecule.
Proteins tend to be more soluble on the alkaline and on the acidic side of this region.
Globular proteins commonly display very low solubility in pure water.
This solubility can be greatly enhanced by the addition of a low level of salt (low ionic
strength). This is referred to as “salting in.” At intermediate levels of salt, the proteins
commonly display a solubility optimum and then become less soluble again at higher
ionic strength which can cause them to precipitate or “salt out.” In small amounts, the
ions tend to shield the protein molecules from each other by coming between the
opposing charges of different protein residues and preventing their aggregation and
exclusion of water. At very high concentration (salting out conditions), the water
becomes organized around the salt to such a degree that the water is not able to
surround the protein sufficiently, resulting in precipitation. Each protein has varying
degrees of solubility depending on ionic strength of the solution. The proteins in your
crude extract number over a thousand and exhibit a wide variety of solubility profiles.
Figure 1 represents the solubility of
three different hypothetical
proteins. One would be able to
separate these three proteins by
the appropriate addition of
ammonium sulfate. The salt would
first be added to the point where
the square root of the ionic
strength is roughly 2. Under these
conditions, protein A would be
expected to aggregate and come
out of solution. The precipitated
protein can be readily pelleted by
centrifugation. The supernatant
would then contain proteins B and
C but little protein A, and could
then be treated with additional
ammonium sulfate until the square Figure 1: Relationship of solubility of 3 hypothetical proteins
root of the ionic strength was about to ionic strength.
3. Under these conditions, most of
protein B would be predicted to be in the pellet and most of protein C would remain in
the supernatant after centrifugation.

Pg | 38
The activity you will be performing this week represents a fairly standard way of using
ammonium sulfate to purify the cytochrome P450BM3 from your crude extract. Many of
the first enzymes obtained in pure form were essentially purified and crystallized
through repeated application and variation of ammonium sulfate saturation procedures.
Although other salts can be used, ammonium sulfate has the advantage of high solubility
and a divalent ion which contributes to a high ionic strength (100% saturated
ammonium sulfate is 4.1 M at 20C). The salt also seems to have a stabilizing effect on
many proteins. Commercially available purified enzymes are commonly sold as
suspensions of ammonium sulfate and stored with refrigeration but not frozen. These
preparations are often more stable than dried or frozen preparations of the enzyme.
The procedure is often used early in purification to remove heterogeneous material and
reduce volume. Usually an appreciable but small percentage of the enzyme activity is
lost through this purification in order to enhance the fraction of your protein of interest.
The cleaned-up concentrated fraction is generally much more suitable for techniques
such as chromatography and electrophoresis.
One disadvantage in “salting out” procedures is that the salt left over in the fractions
obtained by this method can interfere with subsequent steps. The primary method to
remove the salt is through dialysis. Dialysis involves using a piece of dialysis tubing,
which is a semi-permeable membrane. The tubing has pores that allow small molecules
to pass in and out freely but not large enough to let protein pass through. The
ammonium sulfate fraction is sealed inside the tubing, and the tubing is added to a
buffer that contains lower levels or no ammonium sulfate. Based on simple diffusion,
the salt molecules will flow from a high to low concentration, decreasing the salt
concentration in the protein extract.

Pg | 39
EXPERIMENT 3 - Ammonium sulfate saturation of cell
extract

REAGENTS:
Extraction buffer: 50mM potassium phosphate
10mM EDTA
1mM DTT
0.1mM PMSF
Cell free extract containing cytochrome P450BM3 activity and absorption peak at 418 nm
50ml size Sorvall tubes
10ml plastic graduated cylinder
50 or 100ml glass beaker
stir bar, magnetic stirrer
weighing boats, scale
dialysis tubing

****************************************************************************************
The enzyme fractions you collect during the protein purification process should
always be kept on ice. Many enzymes can have a loss of activity if not kept on ice.
****************************************************************************************

PROCEDURE:
 Members of the group should split responsibilities so all have the opportunity to participate
in purification, Bradford assays, and enzyme activity assays. Save all protein samples for the
Bradford until the end of the purification (so only 1 Bradford needs to be performed).
 Based on your data from previous weeks, choose the fraction that contains your P450. This is
the extract you will use today. Save the other extract as you will use it next week.
 Measure the volume of the cell fraction by decanting it into a graduated cylinder. What is this
volume?
 Aliquot 100l of the extract into a microcentrifuge tube. Label it with (1) cell free extract A or
B (2) group ID and (3) the date. Give the tube to your TA to place in the freezer. This aliquot
will be used next week.
 Place a stir bar in a glass beaker (50 or 100 ml size). Decant the extract from the graduated
cylinder into the beaker. Place the beaker in a dish with ice. Rinse out the graduated cylinder
with distilled water and place it upside down on a paper towel to dry.

Pg | 40
 The precipitation of the proteins in the extract by ammonium sulfate will be done in
increments. The dry powder of ammonium sulfate will be added to the extract in amounts
that will give:
1st: 0-40% (NH4)2SO4 saturation
2nd: 40-60% (NH4)2SO4 saturation
3rd: 60-80% (NH4)2SO4 saturation
 The amount in grams of ammonium sulfate that needs to be added will be calculated by using
the table on the following page. Find the row in the first column for the initial percent of
ammonium sulfate saturation, then move right along that row to the column headed with the
percent of saturation desired. Be aware that this is the amount of ammonium sulfate to add to
one liter of solution. However, the volume you measured in step 2 is much less than one liter
so you need to add a proportionately smaller amount.
 Ex. You have 15ml of extract for the 0-40% (NH4)2SO4 step. The table indicates you want to add
243 grams of (NH4)2SO4 per liter of CFE. So for 15ml you will want to add 3.65 grams of
(NH4)2SO4.
 Determine how many grams of ammonium sulfate you need to weigh out for your first
 ammonium sulfate cut: 0-40%. How much ammonium sulfate will you add?
 Check your calculations with your TA. Weigh out the ammonium sulfate in a weighing boat
on the scale provided.
 Stir the extract gently with the stir bar (2 or 3 on the stir plate dial). Add the ammonium
sulfate you calculated for 0-40% saturation slowly to the beaker. Allow time for the
ammonium sulfate to dissolve after each addition. As you approach 40%, you will see the
extract become cloudy. After the last addition of ammonium sulfate, continue stirring gently
for 10 minutes.
 If you see foam at the surface, you are stirring too rapidly. The frothing is indicative of
protein denaturation. The speed at which you add (NH4)2SO4 is important. As (NH4)2SO4
dissolves, a large amount of water is bound to the (NH 4)2SO4 molecules. So as (NH4)2SO4
molecules in solution increase, less water is available to interact with proteins. If done too
quickly or slowly, it can result in the unexpected denaturation of certain proteins.

Pg | 41
 INITIAL CONCENTRATION OF AMMONIUM SULFATE, % SATURATION FINAL CONCENTRATION OF AMMONIUM SULFATE, %SATURATION

10 20 25 30 33 35 40 45 50 55 60 65 70 75 80 90 100

GRAMS SOLID AMMONIUM SULFATE TO BE ADDED TO 1 LITER OF

SOLUTION

0 56 114 144 176 196 209 243 277 313 351 390 430 472 516 561 662 767

10 57 86 118 137 150 183 216 251 288 326 365 406 449 494 592 694

20 29 59 78 91 123 155 189 225 262 300 340 382 424 520 619

25 30 49 61 93 125 158 193 230 267 307 348 390 485 583

30 19 30 62 94 127 162 198 235 273 314 356 449 546

33 12 43 74 107 142 177 214 252 292 333 426 522

35 31 63 94 129 164 200 238 278 319 411 506

40 31 63 97 132 168 205 245 285 375 469

45 32 65 99 134 171 210 250 339 431

50 33 66 101 137 176 214 302 392

55 33 67 103 141 179 264 353

60 34 69 105 143 227 314

65 34 70 107 190 275

70 35 72 153 237

75 36 115 198

80 77 157

90 79

Table for determining the amount of ammonium sulfate needed per liter to yield various percentages of saturation. A
saturated solution is 4.1 M and 3.9 M in ammonium sulfate at 25 and 0 C, respectively. The initial and final concentrations of
ammonium sulfate are found on the vertical and horizontal scales, respectively. The point of intersection of two lines drawn from
these points indicates the number of grams of ammonium sulfate that must be added to each liter of solution at the initial
concentrations to yield one of the final concentrations. [Modified from Methods of Enzymology, Volume I, Academic Press, New
York, 1968, page 76.]

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  Pour the contents of the beaker into a Sorvall tube and centrifuge at 12,000 rpm for 10
minutes. The beaker and stir bar can be reused in the next ammonium sulfate cut.
 Position the Sorvall tube so the pellet is on top as you pour the supernatant out of the tube,
pour the supernatant into a graduated cylinder. Record the volume: ______
 Pour the supernatant into the beaker with a stir bar and calculate the grams of (NH 4)2SO4 to
add for 40% to 60% saturation. Add (NH4)2SO4 slowly again. Let it stir for 10 minutes after the
last addition. Rinse the graduated cylinder and drain.
 Resuspend the pellet from step 8 with 1ml extraction buffer using an applicator stick. Do not
use a P1000 as it will result in too much foam.
 After mixing, use a graduate cylinder to measure. Record the volume: Aliquot into a
vial(s) and label with: 0-40%, your group number and the date.
 Perform an enzyme activity on the 0-40% (NH4)2SO4 fraction from step 10 (use 3l). Consult
week 2 experiments for the set-up of the assay.
 Pour the contents of the beaker from step 9 into a Sorvall tube and centrifuge at 12,000 rpm
for 10 minutes. The beaker and stir bar can be reused in the next ammonium sulfate fraction.
 Position the tube so the pellet is on top as you pour the supernatant out of the tube, pour the
supernatant into a graduated cylinder. Record the volume:
 Pour the supernatant into the beaker with a stir bar and calculate the grams of (NH 4)2SO4 to
add for 60% to 80% saturation. Add (NH4)2SO4 slowly again. Let it stir for 10 minutes after the
last addition. Rinse the graduated cylinder and drain.
 Resuspend the pellet from step 13 with 1ml extraction buffer using an applicator stick. Do not
use a P1000 as it will result in too much foam.
 After mixing, use a graduate cylinder to measure. Record the volume:
 Perform the enzyme activity assay on the 40–60% fraction (use 3 l).
 Pour the contents of step 14 into a Sorvall tube and centrifuge at 12,000 rpm for 10 minutes.
 The 80% supernatant fraction can be thrown out.
 Resuspend the pellet from with 1ml extraction buffer using an applicator stick. Measure the
volume with a graduated cylinder and record it
 Assay this 60-80% ammonium sulfate fraction for enzyme activity (use 5 l).
 Perform a Bradford assay on each of your resuspended pellets (0-40%, 40-60%, 60-80%). Look
back at week 2 for information on how to set up the assay. You will also need to set-up a new
set of BSA standards.
 Dilute each ammonium sulfate fraction as you did on your cell free last week (most will be
using a 1:25 dilution). Then use 5l of this dilution for the Bradford.

You should record the Bradford and enzyme activity data from each of the ammonium
sulfate fractions.
Was there a color difference between the fractions? Did this have any correlation with
the results of the enzyme activities?

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Choose the pellet with the highest enzyme activity for future use. To use this fraction,
the ammonium sulfate needs to be removed. Take a piece of tubing from the beaker
filled with water. Tie two knots at one end. Pipette the ammonium sulfate fraction that
has the reddest color either with a P1000 into the open end of the dialysis tube. Tie off
that end with two knots as well. Label an orange dialysis clip with tape and a permanent
pen: (1) ammonium sulfate fraction, (2) group number, (3) and date. Clip onto the end of
the tubing (outside the knot). Place in a 2 liter flask with extraction buffer. The flask will
be stirred overnight in a cold room. The staff will remove your fractions tomorrow and
centrifuge them to obtain only the soluble protein.

FOR THE LAB REPORT:

For the one ammonium sulfate fraction that
you chose for dialysis, fill out Table 2 on the
following page.
Perform the following calculations on the
fraction that you chose for dialysis.

𝐓𝐨𝐭𝐚𝐥 𝐚𝐜𝐭𝐢𝐯𝐢𝐭𝐲 𝐨𝐟 𝐟𝐫𝐚𝐜𝐭𝐢𝐨𝐧

Calculation of %yield = 𝐗 𝟏𝟎𝟎
𝐓𝐨𝐭𝐚𝐥 𝐚𝐜𝐭𝐢𝐯𝐢𝐭𝐲 𝐨𝐟 𝐜𝐞𝐥𝐥 𝐟𝐫𝐞𝐞 𝐞𝐱𝐭𝐫𝐚𝐜𝐭 (𝐛𝐞𝐟𝐨𝐫𝐞 𝐚𝐦𝐦𝐨𝐧𝐢𝐮𝐦 𝐬𝐮𝐥𝐟𝐚𝐭𝐞)

𝐒𝐩𝐞𝐜𝐢𝐟𝐢𝐜 𝐚𝐜𝐭𝐢𝐯𝐢𝐭𝐲 𝐨𝐟 𝐟𝐫𝐚𝐜𝐭𝐢𝐨𝐧

Calculation of fold purification =
𝐒𝐩𝐞𝐜𝐢𝐟𝐢𝐜 𝐚𝐜𝐭𝐢𝐯𝐢𝐭𝐲 𝐨𝐟 𝐜𝐞𝐥𝐥 𝐟𝐫𝐞𝐞 𝐞𝐱𝐭𝐫𝐚𝐜𝐭 (𝐛𝐞𝐟𝐨𝐫𝐞 𝐚𝐦𝐦𝐨𝐧𝐢𝐮𝐦 𝐬𝐮𝐥𝐟𝐚𝐭𝐞)

You will include the Bradford and enzyme activity results for this fraction in your lab
report as well.

Practice Problems (Not for lab report):

 A lab is purifying protein X with an ammonium sulfate saturation. Normally they would expect it to
precipitate in a 30-50% ammonium sulfate solution. While adding ammonium sulfate salt to their
extract for the 0-30% solution, the graduate student accidentally dumps all of the ammonium sulfate in
the tube (rather than slowly adding). What effect will this have on the purification of protein X?

Pg | 44
 Table 2: Protein Purification Chart

_______% to ______%
Ammonium Sulfate
Cell Free Extract Fraction (fraction with Gel filtration extract
(last week’s data) most P450) (will fill in next week)
I
Total extract volume (ml)

II
Volume of extract used in Bradford
assay (l)

III
O.D. from Bradford assay

IV
mg protein used in Bradford assay
(based on BSA standard curve)

V
Extract concentration
IV/(II x 10-3) (mg/ml)

VI
Total mg protein
IxV

VII
Volume of extract used in enzyme
assay (l)

VIII
 O.D. per second
(slope of best fit line)

IX
moles NADPH oxidized per sec.

X
mg protein used in enzyme assay
(VII x 10-3x V)

XI
Specific Activity
moles NADPH oxidized per sec per
mg protein (IX / X)
XII
Total Activity
Specific activity x Total Protein
(XI x VI)

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WEEK 4
A. Further Purification of Cytochrome P450 Extract Through Gel Filtration
Chromatography
B. Polyacrylamide Gel Electrophoresis

Definitions
chromatography:
A process in which complex mixtures of molecules are separated based on certain
physical properties (charge, size, affinity for a given molecule)

eluate:
The solution containing your molecules of interest that is removed from a
chromatographic column.

gel filtration chromatography:

A chromatographic procedure for the separation of a mixture of molecules by size;
based on the capacity of porous polymers to exclude solutes above a certain size.

Polyacrylamide gel electrophoresis (PAGE):

An electrophoretic method in which molecules migrate through a molecular lattice (the
gel) created by polymerized, cross-linked polyacrylamide

Running (or separating) gel:

A gel in which the pores are small enough to have a molecular sieving action, which
serves to separate sample molecules

Stacking gel:
A gel in which the pores are very large so protein motility is not impeded. The proteins
in the well will “stack” into a single band when they hit the running gel.

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Chromatography
Last week you separated your protein of interest, Cytochrome P-450BM3, from some of
the other soluble proteins that are present in E. coli by ammonium sulfate precipitation.
You made 3 fractions with different concentrations of ammonium sulfate: 0-40%,
40-60%, and 60-80%. Which fraction has the reddest color? This red fraction is the one
you will use for this week’s experiment.
Next, you will further separate Cytochrome P-450BM3 from other proteins by
chromatography. A.J.P. Martin and R.L. Synge won the Nobel Prize in chemistry in 1952
for developing the theory and practice of chromatography. Martin defined
chromatography as “the uniform percolation of a fluid through a column of more or less
finely divided substance, which selectively retards, by whatever means, certain
components of the fluid.” So what you will be concerned with today is the efficient
separation of your protein from many of the other proteins in the ammonium sulfate
fraction.
There are many types of chromatography: affinity, gel filtration, ion-exchange,
adsorption, gas liquid (GLC), high performance liquid (HPLC), reverse phase, and
supercritical fluid. The mechanisms of separation differ in each case but the technique
is usually similar for all. In each procedure, one adds the fraction containing the protein
of interest to a column. Then, with a gradient of solute or with a specific ligand, your
protein of interest is eluted from the column and collected in a series of fractions.
Ion exchange chromatography separates proteins on the basis of charge and gel
filtration chromatography, on the basis of size. The procedures depend on small
physiochemical differences of the proteins in the mixture, and the affinity between the
chromatographic matrix (or medium) and the proteins.
You will continue the purification of your protein, Cytochrome P450BM3, by gel filtration
chromatography. While you are only performing gel filtration chromatography, below
are brief descriptions of the main types of chromatography commonly used in
biochemistry labs.

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Affinity Chromatography
Affinity chromatography is the separation of macromolecules on the basis of
biological function. The macromolecule can be an enzyme, an antibody, nucleic acids,
repressor proteins, transport proteins, and receptor proteins, among others. Affinity
chromatography is based upon the principle that the macromolecules bind to a specific
molecule called a ligand (e.g. if the macromolecule is an antibody, then the ligand is an
antigen; if the macromolecule is an enzyme, the ligand can be the substrate). The table
below lists some macromolecules and their ligands.
Purification of various macromolecules using affinity chromatography
Macromolecule Immobilized ligand

Adenosine deaminase Adenosine

Aminopeptidase Hexamethylenediamine
Avidin Biotin
Carbonic anhydrase Sulfanilamide
Chorismate mutase Tryptophan
Chymotrypsin Tryptophan
Glycerol 3-phosphate dehydrogenase Glycerol 3-phosphate
Isoleucyl tRNA synthetase Aminoacyl tRNA

The appropriate ligand is attached to the

matrix of the column and a protein
extract is run through the column. The
only macromolecule that will attach to
the column will be the one that binds to
the ligand; all the other macromolecules
will wash through the column. The
column is then treated in such a manner
that the ligand and the macromolecule
will dissociate and the macromolecule
will then elute off. Elution by inclusion
of free ligand in buffer is common.
Other methods of elution can be
increasing or decreasing salt
concentrations, lowering the pH of
buffer, decreasing the temperature of
the column, detergents, and dilution techniques.

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Ion Exchange Chromatography
In ion-exchange chromatography, proteins bind by electrostatic forces to the column.
There are two types of ion-exchangers: cation exchangers and anion exchangers.
A protein with a net negative charge would tend to bind to an anion exchanger and a
protein with a net positive charge would tend to flow through the anion exchange
column.
The strength of the binding of each protein to the residue of the column is affected by
the strength of the buffer, competing ions in the buffer and the pH of the buffer. Quite
often, gradients of buffer concentrations and pH are used to optimize purification of a
particular protein.

Gel Filtration Chromatography

Gel filtration chromatography has been referred to by several other names: molecular
sieving, size-exclusion or gel exclusion. Gel filtration is a useful method for desalting
protein mixtures or removing smaller proteins and does not interfere with the biological
or enzyme activity of the protein of interest. It is a quick, gentle, and practical method
for separating your protein of interest, cytochrome P450BM3, from contaminating
molecules and small proteins. The chromatographic matrix is composed of spherical
beads containing pores.
When a mixture of different sized molecules (like your ammonium sulfate fraction) is
placed in the gel filtration column, the larger molecules (like Cytochrome P450BM3)
cannot easily diffuse into the pores and are eluted from the column first. The small
molecules diffuse into the pores of the gel beads and will pass through the column much
more slowly. The exclusion limit of the gel is the molecular weight of the smallest
molecule incapable of penetrating gel pores. Your gel filtration column is filled with
Sephadex-G25 for proteins larger than 5kD.

Image from http://www.sci.sdsu.edu/TFrey/Chem365/Proteins/ProteinsChem365.html

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EXPERIMENT 4A - Gel filtration chromatography
REAGENTS:
1 gel filtration column
Ammonium sulfate fraction
equilibration buffer: 100mM potassium phosphate, pH 7.4

PROCEDURE
 Obtain your protein fraction that underwent dialysis last week. Save 5l of this fraction in a
1.5ml tube and place it on ice. This will be used for the protein gel later on.
 Your gel filtration column has been equilibrated with 100mM potassium phosphate, pH 7.4.
Open the stopcock and allow the buffer to drain until the meniscus of buffer is at the bed of
the matrix. Turn off the stopcock. Set up a mini-rack with 12 Eppendorf tubes labeled 1-12.
 Aliquot the ammonium sulfate fraction to the gel filtration column. Be careful not to disturb
the bed of the column matrix. Open the stopcock. Let the meniscus reach the bed of the
column matrix then turn the stopcock off.
 Add the equilibration buffer and open the stopcock. As the buffer migrates down the column,
the red color will become diffuse and you will not see it as a discrete red band. Do not collect
the initial solution as it will not contain any P450 protein.
 Once the red color nears the bottom of the column, start collecting 0.5ml aliquots while
continuously adding equilibrium buffer at the same time. You should be able to see a
gradation in color from slightly red to colorless. Put each tube on ice after collection.
 Decide which 2 or 3 fractions are the reddest by lining the tubes up against a white paper
towel. Consult your TA for advice. Combine the reddest fractions using a micropipettor to
measure the volume of your sample. Record this volume in your protein purification chart on
Table 2 (from the previous week).
 From the combined fractions, aliquot 0.3ml of the gel filtration eluate into the quartz cuvette.
Run an absorption spectrum from 250 to 600nm. After completing the scan, add the aliquot
back with the rest of the protein fraction.
 Do an enzyme assay and Bradford assay. Use 3l of gel filtration column eluate for enzyme
assay and 1l for Bradford assay. These values may have to be adjusted depending on the
results.
 Give your TA the tube containing gel filtration eluates.

Now the purification of cytochrome P450BM3 is complete. The next two weeks will be
spent characterizing the enzyme kinetics of the protein in the gel filtrate.

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Calculations for enzyme activity assays
Next week, you will be running a number of assays on your purified enzyme extract.
You will vary the amount of enzyme and analyze the reaction rates for kinetics studies.
In order to do these assays, you will need to use a certain amount of enzyme. For the
experiment varying concentration of enzyme, you will need to use 0, 25, 50, 75, 100, 150,
and 200 picomoles of enzyme per cuvette.
To prepare for the concentrations of enzyme to use, calculate the volume of extract
needed to obtain the given picomoles of enzyme. The calculations will be based upon
the absorption spectrum data obtained for the gel filtration combined eluate. The peak at
418nm represents the heme group of your enzyme which will be used to figure out
picomoles of enzyme. There is one heme group per enzyme. Picomoles are 10-12 moles.
The extinction coefficient at 418nm is 91,000 M-1cm-1. Show all your calculations.
O.D. of gel filtration eluates at 418 nm
Volume of gel filtration needed to give:

0 picomoles/cuvette l

10 picomoles/cuvette l

20 picomoles/cuvette l

25 picomoles/cuvette l

30 picomoles/cuvette l

40 picomoles/cuvette l

50 picomoles/cuvette l

70 picomoles/cuvette l

100 picomoles/cuvette l

125 picomoles/cuvette l

150 picomoles/cuvette l

Pg | 51
FOR THE LAB REPORT:
For the gel filtrate, fill out Table 2 from last week.
Perform the following calculations as well.

𝐓𝐨𝐭𝐚𝐥 𝐚𝐜𝐭𝐢𝐯𝐢𝐭𝐲 𝐨𝐟 𝐟𝐫𝐚𝐜𝐭𝐢𝐨𝐧

Calculation of %yield = 𝐗 𝟏𝟎𝟎
𝐓𝐨𝐭𝐚𝐥 𝐚𝐜𝐭𝐢𝐯𝐢𝐭𝐲 𝐨𝐟 𝐜𝐞𝐥𝐥 𝐟𝐫𝐞𝐞 𝐞𝐱𝐭𝐫𝐚𝐜𝐭 (𝐛𝐞𝐟𝐨𝐫𝐞 𝐚𝐦𝐦𝐨𝐧𝐢𝐮𝐦 𝐬𝐮𝐥𝐟𝐚𝐭𝐞)

𝐒𝐩𝐞𝐜𝐢𝐟𝐢𝐜 𝐚𝐜𝐭𝐢𝐯𝐢𝐭𝐲 𝐨𝐟 𝐟𝐫𝐚𝐜𝐭𝐢𝐨𝐧

Calculation of fold purification =
𝐒𝐩𝐞𝐜𝐢𝐟𝐢𝐜 𝐚𝐜𝐭𝐢𝐯𝐢𝐭𝐲 𝐨𝐟 𝐜𝐞𝐥𝐥 𝐟𝐫𝐞𝐞 𝐞𝐱𝐭𝐫𝐚𝐜𝐭 (𝐛𝐞𝐟𝐨𝐫𝐞 𝐚𝐦𝐦𝐨𝐧𝐢𝐮𝐦 𝐬𝐮𝐥𝐟𝐚𝐭𝐞)

You will include the Bradford and enzyme activity results for this fraction in your lab
report as well.

Practice Problems (Not for lab report):

 For extra credit, you decide to perform affinity chromatography following the gel filtration
chromatography for your P450 containing extract. Describe the column you would use.

 A student is doing anion exchange chromatography. He accidentally adds concentrated

sodium hydroxide into his protein extract. How might this affect the chromatography?

Polyacrylamide Gel Electrophoresis

This week you will add your protein fractions to a polyacrylamide gel. Two groups will
share one gel to load their protein fractions. After polyacrylamide gel electrophoresis
(PAGE), the gel will be stained with RAPID Stain™ to detect the proteins in the various
fractions remaining following the purification procedure.
Like when used with DNA, the goal of electrophoresis is to maximize the difference in
mobility and the separation of molecules. The rates of migration of proteins depend on
the strength, viscosity, pH, temperature, and chemical nature of the medium in which
the protein is moving. Thus, proteins carrying a net negative charge in a given solution
will migrate towards the anode (positive electrode which attracts anions). Proteins
carrying a neutral or positive charge in the solution will either not migrate or be
attracted to the cathode (negative electrode which attracts cations), respectively. Such
attraction and repulsion creates differential migration rates. As for the molecular weight

Pg | 52
(size) of proteins, the smaller the molecular weight, the faster the protein migrates
through the gel, and vice-versa.
You will boil your protein extract in 2x sample buffer containing sodium dodecyl sulfate
(SDS) and -mercaptoethanol (BME). This results in total disruption of protein
complexes and denaturing of proteins. The BME reduces all disulfide bonds in the
protein, which helps (along with the heating) to unfold the protein. SDS is an anionic
detergent, which coats the denatured protein, conferring a fairly uniform negative
charge to the protein. All SDS-coated polypeptides assume rod-like shapes with
dimensions proportional to their molecular weights. Therefore, the proteins travel
towards the anode with differential rates based primarily on size, without interference
by variations in shape and overall charge.
The gel used today is polyacrylamide. Polyacrylamide is made of two monomers:
acrylamide and N,N-methylene bisacrylamide (known as bis). These two monomers
crosslink into a polymer when mixed together in the presence of two compounds,
ammonium persulfate (APS) and N,N-tetramethylenediamine (TEMED). APS is a
powerful oxidizing agent and can undergo a one-electron reduction:

S2O82- + 1e-  SO42- + SO4-

SO4- is a free radical and is the initiator for the polymerization of acrylamide. The
decomposition of APS is promoted by TEMED.
Note: Cytochrome P450BM3 is a 119KDa protein.
The linking of the acrylamide monomers into polyacrylamide creates pores, which have
a sieving effect in hindering proteins according to size as they are pulled through the gel
by the electrical force. The concentration of acrylamide and the ratio of acrylamide to
bis determines the pore size. Increasing either reduces pore size and slows the
movement of larger proteins.
The polyacrylamide gels used in class today are formed in 2
sections. The bottom part of the gel, called the separating (or
resolving or running) gel, occupies about 75% of the gel and is 10%
acrylamide. The top of the gel, called the stacking gel, occupies the
upper 25% of the gel, includes the wells where the protein sample
is added and is 4% acrylamide. The stacking gel has large pores,
which do not impede the movement of proteins. The proteins in
the stacking gel rapidly "stack" into a very narrow region and
move through the stacking gel as very narrow individual zones. In the separating gel,
the proteins enter as a series of very thin closely spaced bands. The separating gel has
small pores and the proteins "unstack" and are separated on the basis of their size.
In the electrophoretic set-up you will use today, the gel is mounted vertically between 2
buffer chambers each containing a separate electrode (anode is red and cathode is
black). The only connection between the chambers is through the gel. When an

Pg | 53
electrical field is applied, the charged proteins will migrate from one end of the gel
(cathode) to the other (anode).
You will be able to monitor how long the gel has been running by watching the
migration of a blue dye, bromophenol blue, which has been added to the 2x sample
buffer. It is not a size marker, but an indicator of how far a gel should run. The dye
migrates with the ion front and will run rapidly through the gel. When it approaches the
bottom of the gel, the run is complete.
After the electrophoresis run, the proteins are fixed and stained by immersing the gel in
an acidic/alcoholic solution of RAPID Stain™. RAPID Stain™ contains Coomassie dye
which only stains protein on a polyacrylamide gel. The molecular weight of the
protein(s) of interest is deduced by comparing their migration relative to the rainbow
marker.

EXPERIMENT 4B - SDS-PAGE
Reagents:
Polyacrylamide gel already mounted in tank (2 gels for 1 tank)
Electrophoresis gel unit with power supply
30% acrylamide/bis solution
Stacking gel: 1.1ml water
2.5ml 0.5M Tris-Cl, pH 6.8
100l 10% SDS
1.3ml 30% acrylamide/bis soln
50l 10% ammonium persulfate
10l TEMED
Separating gel: 2.5ml 1.5M Tris-Cl, pH 8.8 4.05 ml water
100l 10% SDS
acrylamide/bis soln
75l ammonium persulfate (10%)
7.5l TEMED
Tank Buffer
2X sample buffer: 0.125M Tris-Cl, pH 6.8
4% SDS
20% glycerol
captoethanol (BME)
0.02% bromophenol blue
Protein samples: Cell free extract A
40-60% (NH4)2SO4 cut (or whichever had the most P450)
Gel filtration eluate
Cell free extract B

Pg | 54
 Heating block set at 100o C
Micropipettor with gel loading tips
Razor blade
RAPID Stain™

Procedure:
 Make sure heating block is on and set at 100o C.
 On the SDS-PAGE for the P450 gel, you will load the following protein fractions: Cell Free
Extract A (wells 1&7), Cell Free Extract B (wells 2&8), 40-60% (NH4)2SO4 cut (wells 3&9), and
gel filtrate (wells 4&10).
 Label 4 microcentrifuge tubes: A, B, 40-60%, and gel filtrate. Don’t forget to also label with
your group number.
 The suggested dilutions are in the table below. However, be aware these dilutions may not fit
your particular fraction – consider how much extract you have been using these past weeks
compared to what the manual suggests. Consult your TA if you have questions.
 Make necessary dilutions in labeled microcentrifuge tubes for each fraction (total volume
should be 50µl).
Gel – One group will use their samples to load lanes 1-5, the second group will load 6-10

Wells Sample to load

20l of 1/2 dil. Of Cell Free Extract A

1&7 (25l CFE-A, 25l H2O, 50l 2X Sample Buffer)
20 l of 1/3 dil. of Cell Free Extract B
2&8 (17l CFE B, 33l H2O, 50 l 2X Sample Buffer)
20 l of 1/10 dil of 40-60% cut
3&9 (5l of 40-60% cut, 45l H2O, 50l 2X Sample Buffer)
20 l of gel fil
4 & 10 (10l of gel filtrate, 40l H2O, 50l 2X Sample Buffer)
15 l of rainbow marker
5&6 (TA will load this)

 Add 50µl of sample buffer to each tube and mix. Remember to change pipette tips between
each sample. The final volume in each tube should be 100µl.
 Make sure the caps on the tubes are tightly closed. Place tubes in the 100o C heating block for
4 minutes. Briefly spin in the centrifuge to remove the condensation from the top of the tube.

Pg | 55
 See the diagram below to determine how you will load your gel. Load 20l of the protein
sample into each well. Remember to use fresh tips with each sample. Unlike the agarose gel,
the wells on the acrylamide gel are difficult to see. Your TA will load 15l of rainbow marker
into well 1.

 After loading gel the, place the lid on the tank, adjust the voltage to 200 volts and run the gel
until the tracking dye is about I cm from bottom of gel. This should take 45 minutes to an
hour. Turn off the power before handling the gel apparatus.
 Wear disposable gloves for this part. Lift the gel holder assembly from the buffer tank. Open
the two levers on the gel holder assembly as you remove it slowly from the tank. The two gels
will fall away from the gel holder assembly and the running buffer will pour back into the
tank.
 Now remove the gel from the plates as described below:
o White tape on the sides cover a ridge. Use a razor blade to cut the gel along this ridge
which will be on the outside edge of the white tape on both sides of the shorter gel
plate. The gel will be sandwiched between a long plastic plate and a short glass plate.
Remember, the gel is very thin and fragile!
o Wedge a spatula between the plates and slowly pry them apart. Wet the gel using a
squeeze bottle containing water.
o Use the short glass plate to cut the gel if it sticks to the long plastic plate, or use the
solid edge of plastic comb if the gel sticks to short glass plate. You need to divide the
gel vertically into 2 halves: one half will have Lanes 1 through 5 and the other half will
have lanes 6 through 10. You might be able to see color from the rainbow markers and
use this as a guide for cutting the gel in half. Each group will stain their own half.

 Place your gel into a dish and label the dish with your group number and date.
 Wash the gel three times with water; five minutes for each wash.

 After the third wash, remove all water from the gel. Pour in enough RAPID Stain™ to cover
the gel. Gently shake the gel in stain for 30 minutes. If the protein bands are of sufficient
intensity, pour off the stain into a waste bottle and wash the gel 2 times with water for 10
minutes each. Depending on how much protein is in your sample, bands may be visible
before 30 minutes, in which case you can start your washes earlier.

Pg | 56
  After washing, keep the gel in water.
Take a photo of the gel with your phone and make sure everyone in your group receives a
copy.
RETURN ALL PROTEIN FRACTIONS TO TA FOR STORAGE. DO NOT THROW
AWAY ANYTHING!

The protein standard is referred to as the rainbow marker and consists of 10 proteins
that are artificially colored. As the gel is running you should be able to see these colors.
Below are the molecular weights of the colored markers. Compare the proteins you
identify on your gel to these standards to estimate their molecule weights.

Molecular Weight (Kd) Color

250 blue
160 red
105 green
75 yellow
50 purple
35 blue
30 orange
25 green
15 blue
10 red

FOR THE LAB REPORT:

Include a picture of your gel. Label the band that corresponds to cytochrome P450BM3.

Pg | 57
WEEK 5
A. Effect of Enzyme Concentration on the Rate of the Reaction
B. Determine Km Values for Lauric Acid and Myristic Acid

Definitions
Km:
The substrate concentration that gives half the maximum rate of reaction
Vmax:
The maximum rate of the reaction
turnover number:
The number of times an enzyme molecule transforms a substrate molecule per unit of
time, under conditions giving maximal activity at substrate concentrations that are
saturating

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Determination of Km and Vmax
In the presence of NADPH and O2, Cytochrome P450BM3 catalyzes the hydroxylation of
long chain fatty acids like lauric acid and myristic acid. Electrons are transferred from
NADPH, to the P450 protein, which contains the prosthetic groups, FAD, FMN, and
heme.

Effect of Enzyme Concentration on Reaction Rate

In an enzyme-catalyzed reaction, the enzyme (E) can reversibly combine with the
substrate (S) to form an intermediate complex of enzyme and substrate (ES). This
complex (ES) is converted to product (P) and free enzyme (E). For simplicity, let’s
consider the reaction S  P. The reaction rate is the amount of S reacted (consumed) in
a unit of time or the amount of P formed in a unit of time. Therefore:

The rate of an enzymatic reaction is the velocity at

which the enzyme converts substrate to product
over a given time period. The concentration of
enzyme and substrate are two factors that strongly
influence the enzymatic rate.
A given reaction rate is determined by looking at
the slope of the tangent to a curve on a product
versus time graph (to the right). Thus, we focus on
the linear portion of this curve, the initial rate of
the reaction. Eventually, the rate of the reaction
will decrease either because the reverse reaction
becomes relevant or the activity of the enzyme is affected by the lack of substrate.
Please note that if we measure the reaction rate as “substrate consumed over time”
instead, the graph shown to the right will be different. Plot it!
Working with the enzyme invertase, Michaelis and Menten obtained the following rate
versus substrate concentration graph:

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First order kinetics occurs when the formation of
product is directly proportional to substrate
concentration. Zero order kinetics is when the
formation of product is independent of substrate
concentration. Zero order reaction conditions are ideal
for studying enzyme activity, since the reaction rate
will reflect the properties of the enzyme and not the
substrate.
Enzyme Kinetics
Catalyzed reactions have a number of interesting
kinetic properties, which are not shared by ordinary
chemical reactions. It is these properties specific to
catalyzed reactions that allow life to sustain itself.
Enzymatic reactions display the phenomenon of
saturation with substrate. At very low substrate
concentrations, the rate of an enzymatic reaction
increases by increasing the concentration of v
substrate. However, eventually a point is reached
where the enzyme is working as fast as it can and
further increasing the substrate concentration has
no effect on the reaction rate. Saturation can occur
at concentrations as low as 10-5 M. [S]
The above figure is the graphical representation of saturation. The simple interpretation
of this observation was pointed out by Michaelis and Menten. They argued that an
enzyme (E) reacts with a substrate (S) in the following manner:

k1 k2
E+S ES E+P
k-1 k-2

All the reactions are reversible, but by restricting our observations to the early stages of
the reaction, k-2 will not be involved since the concentration of product (P) will be small.
Saturation occurs because E + S is an ordinary bimolecular reaction and if we increase S,
we convert more of E into ES. Eventually all the E will be present as ES and the rate of
reaction will be at a maximum. Thus, Vmax (maximum rate) will be equal to k2 [ES]. A
hallmark of saturation conditions is that the concentration of ES equals the
concentration of E initially added to the reaction.
When the reaction is under zero order conditions and the enzyme is saturated with
substrate, v = k2 [ES]. The concentration of ES depends on the rate of ES formation and
the rate of ES disappearance.

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The rate of formation of ES is given by the equation:
d [ES]
=k 1 [E][S]
dt

But once the reaction begins, it is difficult to determine how much of the enzyme
initially added is free enzyme (E) or enzyme bound to substrate (ES). All we know is the
amount of enzyme initially added to the reaction, designated as [Etotal] or [ET]. Using this
nomenclature, the amount of unbound enzyme, or [E] in the equation above can be
written as ([ET] - [ES]).
So the rate of formation of ES is:
d [ES]
=k 1 ([ET ] - [ES])[S]
dt

Remember, we are looking early in the reaction, so the k-2 reaction does not contribute
to ES formation.
The rate of disappearance of ES is given by the equation:
d [ES]
− =k -1 [ES] + k 2 [ES]
dt

To integrate these two equations, we must assume that the reaction is at steady state.
What this means is that the rate of ES formation is equal to the rate of its disappearance.
This is a relatively safe assumption at saturation, as [ES] is fairly constant under these
conditions. Thus, the amount of ES disappearing must equal the amount being formed.
With this assumption:
d [ES] d [ES]
− = 𝑎𝑛𝑑 k -1 [ES] + k 2 [ES] = k 1 ([ET ] - [ES])[S]
dt dt

Rearranging this:

([ET ] - [ES])[S] k −1 + k 2
= = Km
[ES] k1

Km is the Michaelis-Menten constant.

What makes the Km a useful constant? If we know the Km of an enzyme for a certain
substrate, we can calculate the proportion of the maximum enzymatic velocity at any
given concentration of substrate. Km values have the units of concentration and a low
Km value (for example 1 x 10-5 M) means that the enzyme has a high affinity for that
substrate. This enzyme will very efficiently convert substrate to product even when the
substrate concentration is low.
There are two basic equations and related graphs (that we will discuss later) that can be
used to determine Km.

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Now recall that the initial reaction rate v = k2 [ES]. Using the equation to derive the
Michaelis-Menten constant above, we can solve for [ES].
([ET ] - [ES])[S]
= Km OR: [ET ][S] - [ES][S] = [ES]K m
[ES]

Rearranging: [ET ][S] = [ES](K m + [S])

ET [S]
So: [ES]=
K m +[S]

This can then be plugged into the initial rate reaction to give:
k 2 ET [S]
v=
K m + [S]

When the enzyme is saturated and all enzyme is bound to substrate, the equation
v = k2[ES] can be rewritten as Vmax = k2[ET], which can then be plugged into the initial
rate equation to give:
Vmax
v=
K m + [S]

This is also known as the Michaelis-Menten equation.

This Michaelis-Menten equation demonstrates that when the substrate concentration
[S] is the same as the Km (remember the units of Km are concentration), v = ½Vmax.
The corresponding graph of rate (v) v. [S] is known as a Michaelis-Menten plot.

The problem with determining the Vmax and Km with a Michaelis-Menten plot, is that
Vmax is only an estimation of the asymptote of the curve. To obtain the actual asymptote
would require an infinite amount of substrate, which is not feasible in the laboratory.

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By rearranging the Michaelis-Menten equation,
another useful equation is obtained, the
Lineweaver-Burk equation which is graphed
1 1
on a Lineweaver-Burk plot ( v vs. [S]
)

The advantage of this plot is that Vmax can be

determined directly from the experimental
data.

EXPERIMENT 5A - Effect of enzyme concentration on the

rate of the reaction
REAGENTS:
100mM potassium phosphate, pH 7.4
10mM sodium laurate in 50 mM potassium carbonate
10mM NADPH
gel filtration eluate

PROCEDURE
 Keep the gel filtrate eluate on ice at all times.
 Perform the enzyme activity assay as in past weeks, except you will vary the amount of gel
filtration eluate. The amounts to be used are 25, 50, 100, and 150 picomoles. You calculated
the volumes needed through the absorption spectrum scan last week. Lauric acid and
NADPH concentration will remain the same (0.1mM). Fill out the chart below.

Cuvette Picomoles Gel Lauric acid NADPH PO4 buffer Total

of gel filtration (ml) volume
filtration eluate (l) (l) (ml)
eluate (l)

1 (blank) - - - - 1.0 1.0

2 100 0.0 1.0

3 25* 1.0

4 50 1.0

5 100 1.0

6 150 1.0
* If your extract is very red, you may want to try 10 picomoles. Consult your TA.
 Incubate the reaction mixture (except for NADPH) at 25C for a few minutes.

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  Cuvette #1 is the blank. After blanking the spectrophotometer, add NADPH, put parafilm on
top of the cuvette and mix. Because of the rapid nature of the reaction, it is advisable to assay
one tube at a time until you are confident enough to try more.
 Cuvette #2 will indicate the OD of the starting concentration of NADPH. Since no substrate is
added what do you expect to see?

Plot your data (O.D. versus time) or have the spectrophotometer provide you with a
graph of the absorbance values. Decide which concentration of enzyme gives the
straightest line over the 2 minute period. Consult with your TA and use this
concentration for the following experiment.
If you do not see a straight line, dilute your enzyme and try again. You will want to see a
decent change in rates (a decent change would be 0.05 O.D. every 20 seconds, 0.01 O.D.
is not).

EXPERIMENT 5B - Determine Km values for lauric acid and

myristic acid

REAGENTS:
100mM potassium phosphate, pH 7.4
10mM NADPH
10mM sodium laurate
10mM sodium myristate (do not leave on ice, if cloudy run the tube under hot water)
gel filtration eluate

PROCEDURE:
 Keep the gel filtrate eluate on ice at all times.
 This exercise deals with kinetics. You will be assaying enzyme activity at various
concentrations of 2 fatty acids, lauric acid, and myristic acid, using a constant amount of
enzyme. You will then calculate Km and Vmax for each substrate used.

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 Fill out the table below. Each reaction will need:
o gel filtration eluate (the concentration deduced from exercise A today)
o 6 concentrations of lauric acid plus the blank
o NADPH (final concentration 0.1 mM)
o PO4 buffer (bring the final volume to 1.0 mls)

FINAL GEL
CONCENTRATION LAURIC FILTRATION TOTAL
OF LAURIC ACID ACID NADPH ELUATE PO4 BUFFER VOLUME
CUVETTE (M) (l) (l) (l) (ml) (ml)

1
(BLANK) - - - - 1.0 1.0

2 0 0 10 1.0

3 25 1.0

4 50 1.0

5 75 1.0

6 100 1.0

7 150 1.0

 Incubate the cuvettes at room temperature for a few minutes before adding NADPH. Use
cuvette #1 as a blank.
 Due to substrate limitation some of the reactions can be completed in less than two minutes.
So you need to move quickly when adding NADPH and placing the cuvettes into the carrier in
the spectrophotometer. Follow the suggestions below.
o Check that the spectrophotometer is set on the correct program and the visible lamp is
on.
o Make sure you start reading the contents as soon as you mix the cuvette.
o Cuvette #2 contains enzyme, NADPH, buffer, but no substrate. So the O.D. reading will
indicate what the O.D. is for 0.1 mM NADPH. What should the plot of this cuvette look
like?
o Cuvettes 3 through 7 should show increasing rates for oxidation of NADPH as all the
necessary components are present. However, it should be stressed that you need to
move quickly to add NADPH to the cuvette, mix by placing parafilm on top and placing
into the carrier in the spectrophotometer. This manipulation should take less than 20
seconds.
o Pay attention to reaction rates. All cuvettes should start at a similar OD.

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  Repeat the above experiment with myristic acid as the substrate. Use concentrations of 0, 20,
40, 60, 80, and 100 M. Add myristic acid right before NADPH addition, as this substrate
tends to precipitate. Lastly, add NADPH.

Protocol Chart
CUVETTE FINAL MYRISTIC NADPH GEL PO4 BUFFER TOTAL
CONCENTRATION ACID (l) FILTRATION (ml) VOLUME
OF MYRISTIC (l) ELUATE (l) (ml)
ACID (M)

1 - - - - 1.0 1.0
(BLANK)

2 0 0 10 1.0

3 20 1.0

4 40 1.0

5 60 1.0

6 80 1.0

7 100 1.0

Before leaving lab, it is important that you briefly analyze the results to ensure that
they make sense. Are the initial readings for each cuvette similar? Does the rate seem to
increase as more enzyme is added?
If the data looks odd, you can still repeat reactions today. If you realize a week or two
from now that the data is bad, there is nothing you can do, which will make assembling
your lab report much more difficult. So it is highly recommended that you take the time
to gather useable data today.
Once you have your data, it is possible to calculate Km and Vmax for both substrates by
graphing the results.
To generate the Michaelis-Menten and Lineweaver-Burke plots, you will need to
determine the rate for each substrate concentration used. Each substrate concentration
will give you a point on the Rate v. [S] or 1/Rate v. 1/[S] graphs. This means for each of
your substrates, you will be graphing 5 different OD v. time curves (cuvettes 3-7).
When determining the rate from the slopes of these lines, be sure that your best-fit line
accompanies a linear reaction. If the reaction is no longer linear towards the end of the

Pg | 66
two minutes, eliminate the non-linear points and generate a new best-fit line.
Incorrectly determining the rate at a specific substrate concentration will negatively
affect your Km and Vmax calculations.

Values That Must Be Included in the Lab Report

 Calculate Km and Vmax for lauric acid and myristic acid.
When you calculated reaction rate, you expressed the enzyme activity in terms of moles of
NADPH oxidized/second. For each substrate concentration (lauric acid and myristic acid),
you need to plot OD versus time, obtain a slope, and calculate moles NADPH oxidized/sec
(v). The 5 substrate concentrations will be [S]. Determine the Km and Vmax via the following
plots:
o Michaelis-Menten plot (v versus [S])
o Lineweaver-Burk plot (1/v versus 1/[S])
Include your OD v. time graphs in the lab report. For each substrate, you should include one
graph with 5 lines (cuvettes 3-7). It is not necessary to have 5 separate OD v. time graphs for
each substrate concentration.
Calculate turnover number of cytochrome P450BM3 (using Vmax of Lineweaver-Burk
plot).
Turnover number is the number of moles of substrate transformed per time per mole of
enzyme (moles NADPH oxidized per second per mole of enzyme). Units are sec-1.

If your Vmax is already in terms of moles as opposed to a concentration (M), then you
can divide by the moles of enzyme added as opposed to the enzyme concentration.
Values of turnover number can range from 50 to about 107 min-1. kcat is also called the
turnover number.

Practice Problems (Not for lab report):

 What are the assumptions we make when deriving the Michaelis-Menten equation?
 TRUE or FALSE. The Km derived from a Michaelis-Menten plot is an estimate. Explain.

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WEEK 6
A. Determination of [I]50
B. Increasing Substrate Concentration to Observe the Effect on Percent Inhibition
C. Determination of Ki

Definitions
Ki:
Dissociation constant of the enzyme-inhibitor complex.

Competitive inhibitor:
Competes with substrate to form an enzyme-inhibitor complex and can be reversed by
increasing substrate concentration. Ki = enzyme-inhibitor dissociation constant.

Noncompetitive inhibitor:
Reacts reversibly with both free enzyme and enzyme-substrate complex; effect is not
reversed by increasing substrate concentration.

Last week you gained some experience with steady state kinetics, estimating Km and
Vmax values for two fatty acid substrates from initial rates. This week we are asking you
to help us begin a systematic investigation of the specificity of inhibition of P450BM3.

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Kinetic Patterns of Enzyme Inhibition
There are two patterns of enzyme inhibition you may observe in lab: competitive and
noncompetitive.
In the presence of these inhibitors, Km and Vmax may be different from the same reaction
in the absence of the inhibitor. These altered values are known as the apparent Km or
apparent Vmax. The relationship between the apparent and actual Km or Vmax is derived
through the factor .

 = 1 + [I]/Ki where [I] is the concentration of inhibitor used in the reaction and
Ki is the dissociation constant.
Ki is a measure of the dissociation of the enzyme-inhibitor complex that describes the
inhibitory strength of a particular molecule. In the reaction below:
E + I  EI
[E][I]
Ki =
[EI]

Ki has the units of M. If the Ki for a reaction is low, the enzyme’s affinity for the inhibitor
is high and vice versa.

Competitive Inhibitor
As the name implies, competitive inhibition involves an inhibitor, which interacts
reversibly with the enzyme’s active site. Thus, the inhibitor competes with the
substrate for position at the active site. The substrate and inhibitor are mutually
exclusive; when the substrate is bound to the enzyme the inhibitor cannot bind and vice
versa. The reaction for competitive inhibition look like this:

At high concentrations of substrate, the substrate will outcompete the inhibitor for the
enzyme’s active site. From the equation, we can see that once the [ES] complex is
formed, product is released without inhibition. Therefore, in competitive inhibition, the
Vmax (when substrate is saturating) for the reaction is unaffected.

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On the other hand, the inhibitor is stealing free enzyme molecules, pushing the
equilibrium backwards (less ES formed). This means more substrate needs to be added
to get the same [ES] in the absence of inhibitor. So, the apparent Km increases. This is a
hallmark of competitive inhibition and can be seen on a Lineweaver-Burk plot:

Notice that the Vmax is the same with or without inhibitor. However, Km has changed
(remember Km is a function of substrate binding). The formula to find Ki is derived as
follows:
where -X is the value of the intercept on the negative
side of the 1/[S] axis and [I] is the inhibitor
concentration.

[𝐈]
Thus: 𝐊 𝐦 𝐚𝐩𝐩 = 𝐊 𝐦 (𝟏 + ) Since all values are known in this equation except Ki, it
𝐊𝐢
is possible to plug in the values of X, Km, and [I] and
solve for Ki.

Noncompetitive Inhibitor
In noncompetitive inhibition, the substrate and inhibitor bind reversibly, randomly,
and independently at different sites: I binds to E and ES; S binds to E and EI. The
resulting ESI complex is inactive.
As a result, Vmax in the presence of an inhibitor is less than Vmax in its absence, because a
proportion of the enzyme will remain in the nonproductive ESI complex. In this case, it
is not possible to outcompete the inhibitor with increasing substrate concentration.
The Km value, on the other hand, will be unchanged because both the free enzyme and
enzyme inhibitor complex have equal affinities for the substrate.

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The reaction for noncompetitive inhibition is the following:
From this mechanism, two Ki constants can be generated,
but because the affinity between the inhibitor and E or ES is
the same, KiEI = KiESI.
[E][I] [ES][I]
K EI
i = and K ESI
i =
[EI] [ESI]

The procedure to find Ki for noncompetitive inhibition is similar to that used to find Ki
for competitive inhibition.

𝑽𝒎𝒂𝒙
𝑽𝒎𝒂𝒙 𝒂𝒑𝒑 =
𝟏 + [𝑰]/𝑲𝒊

Analysis of Ki Data
The purpose of this exercise is to determine the Ki (referred to as the inhibitor constant)
of imidazole or 4-phenylimidazole for P450BM3. Both are classic inhibitors of P450
enzymes and work by binding to the oxygen-binding site. The physical significance of
the Ki depends on the type of inhibition observed.
To determine the type of inhibition you are observing, first complete the three separate
experiments on the following pages. Upon completion of the third exercise, it is
recommended that you carefully observe your data to determine whether it is
necessary to repeat any of the experiments.
Based on the absorbance values, you can determine the general effect that the inhibitor
has on the reaction rate. Later when you graph your data, compare your plots with the
competitive or noncompetitive patterns presented in this text.

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With a competitive inhibitor, increasing the concentration of substrate should decrease
percent inhibition, as observed in the second experiment. This also means the Vmax
calculated from the Lineweaver-Burk plot should be unaffected.
Noncompetitive inhibitors affect the Vmax of the enzyme but have no effect on the
binding of the substrate. With a noncompetitive inhibitor, the Ki value should be
approximately the same as the [I]50 calculated in the first exercise, and it should give a
horizontal line in the plot of the second exercise.
There are also a number of potential experimental errors that could explain why your
data may not fit the patterns described above. Besides experimental error, there could
be issues with how your Lineweaver-Burke plot was created. In preparing your
double-reciprocal plots, make sure your best-fit line is in fact a best-fit line. This may
require you to eliminate some of the latter data points in a reaction, to ensure that your
line is not being fit to a curve. It is also possible that there were errors in one or more of
the reactions. If one data point is way out of line with the others, there probably was
something wrong with that reaction, and you should disregard it.
Remember that you determined the Km of the enzyme with lauric acid during the past
laboratory session. Compare the results between week 5 and 6 for consistentcy. If the
data from week 6 is difficult to analyze, it may be necessary to substitute it with your
week 5 data. Since this was the first week performing the experiment with inhibitor, last
week’s experiment will not help you with the inhibitor reactions.
If you are still confronted with a confusing set of data points either in the presence or
absence of the inhibitor, be sure to examine your data and suggest in your report what
kinds of procedural or technique errors might have been responsible for your inability
to measure the initial rates of your reaction with sufficient accuracy.

EXPERIMENT 6A – Determination of [I]50

Adjust the concentration of
inhibitors so that you get an
inhibition well over 50%, another
inhibition well below 50%, and
another in between these two
values. You only need three
consistent values. Make a plot of
the percent inhibition versus the
log of the inhibitor
concentration:

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Keep in mind, some points on your graph may have a negative X value. Estimate the
[I]50 of your inhibitor by extrapolation on the log plot graph. The [I]50 is the
concentration of inhibitor which should give 50% inhibition.

REAGENTS:
100mM Imidazole in 100mM potassium phosphate, pH 7.4 OR
30M 4-phenylimidazole in 100mM potassium phosphate, pH 7.4
10mM sodium laurate in 50mM potassium carbonate
10mM NADPH
gel filtration eluates

PROCEDURE:
Today you will perform a series of assays using up to three cuvettes in a single run.
Practice and develop your technique as you proceed.
 Fill in the blanks of the chart below with:
o Varying inhibitor volumes
o P450BM3 gel filtration (same volume you used last week for the K m experiment)
o 100mM potassium phosphate buffer, pH 7.4

CUVETTE SUBSTRATE NADPH INHIBITOR GEL PO4 TOTAL

(LAURATE) (l) (l) FILTRATION BUFFER VOLUME
(l) ELUATE (ml) (ml)
(l)
1 (blank) - - - - 1.0 1.0

2 10 10 0.0 1.0

3 10 10 5 1.0

4 10 10 10 1.0
5 10 10 15 1.0

 Contrary to the previous weeks’ assays, you will add the enzyme last, not the NADPH. Add all
reagents except enzyme and mix. Incubate cuvettes at 25C for a few minutes.
 Blank the machine. To start the reaction, add the enzyme to the cuvettes and immediately
insert into the spectrophotometer. This may be easiest if you place all cuvettes in the holder
prior to adding the enzyme, and tilting the entire holder to keep the enzyme drop from
mixing with the rest of the reaction.
 Upon completion of the reaction, obtain the rate of the reaction from the
spectrophotometer. It is not necessary to graph the reactions for this exercise.

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Collect your data and determine the percent inhibition with each amount of inhibitor.
Slope of rate observed 𝐰𝐢𝐭𝐡 inhibitor
(1 − ) X 100
Slope of rate observed 𝐰𝐢𝐭𝐡𝐨𝐮𝐭 inhibitor

EXPERIMENT 6B – Increasing substrate concentration to

observe the effect on percent inhibition
We would now like to determine whether your inhibitor follows a competitive pattern or
noncompetitive pattern. As a first step, we want to see if the percent inhibition changes
as you increase the substrate under constant inhibitor concentrations. In
noncompetitive inhibition, the percent inhibition remains constant as we change the
substrate concentration. In competitive inhibition, the percent inhibition decreases as
the substrate concentration is increased or vice versa). We will now determine which
situation applies to our inhibitors by plotting the inhibition at 3 different substrate
concentrations with each of your inhibitors (see figure below):

You will use the [I]50 for the inhibitor estimated in the previous experiment, which
should give us the central point in the desired plot. Set up controlled assays with
inhibitor at the estimated [I]50 concentration and the substrate concentration at one half,
at the same and at twice the level of the standard substrate concentration used in the
previous assays.

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Complete the table below for the aliquots of the reagents you will be adding. The amount
of inhibitor to add was determined from the first exercise this week. The amount of gel
filtrate to add was determined last week (and used on today’s first experiment).
Calculate the percent inhibition and plot as shown for each inhibitor. Again, feel free to
obtain the rates of each reaction from the spectrophotometer.

CUVETTE LAURIC NADPH INHIBITOR GEL PO4 TOTAL

ACID (L) (L) FILTRATION BUFFER VOLUME
(L) (I 50 CONC) ELUATE (ml) (ml)
(L)

1 (blank) - - - - 1.0 1.0

2 5 10 0.0 1.0

3 5 10 1.0

4 10 10 0.0 1.0

5 10 10 1.0

6 20 10 0.0 1.0

7 20 10 1.0

If there is no appreciable effect of substrate concentration on the inhibition, a

noncompetitive inhibition is indicated. In that case, the Ki for the inhibitor (which will
be determined in the third exercise) should be close to the [I]50 you calculated in the
previous exercise.
If the percent inhibition decreases with increasing substrate concentration, the
inhibition could correspond to the classical competitive pattern. With a competitive
inhibitor, the Ki does not correspond to the [I]50 and can be calculated in the third
exercise to follow.

EXPERIMENT 6C – Determine the Ki

Repeat the determination of the Km you made with lauric acid last week. In addition, you
will perform the same set of reactions in the presence of the inhibitor. Remember to add
the enzyme last.

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Fill in the two protocol tables below with the proper aliquot amounts:
REPEAT OF Km

CUVETTE FINAL LAURIC NADPH GEL PO4 TOTAL

CONCENTRATION ACID (L) FILTRATION BUFFER
OF LAURIC ACID (L) ELUATE (ml) VOLUME
(M) (L) (ml)

1 (blank) - - - - 1.0 1.0

2 25 10 1.0

3 50 10 1.0

4 75 10 1.0

5 100 10 1.0

6 150 10 1.0

Ki

CUVETTE FINAL LAURIC NADPH GEL FILT. INHIBITOR PO4 TOTAL VOL.
[LAURIC ACID (L) ELUATE (~I50 CONC. = BUFFER (ml)
ACID] (L) (L) _____) (L) (ml)
(M)

1 (blank) - - - - - 1.0 1.0

2 25 10 1.0

3 50 10 1.0

4 75 10 1.0

5 100 10 1.0

6 150 10 1.0

 Add the proper aliquots of lauric acid, inhibitor, NADPH, and bring up to 1.0ml volume with
phosphate buffer. Incubate the ingredients for a couple minutes before adding the enzyme
(last!).
 Use the program on the spectrophotometer for 2 (or 3 or 4 if you’re up to it) cuvettes. You
need to add the enzyme quickly and put in the cuvette carrier as quickly as possible.

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FOR THE LAB REPORT:
 Graph the % inhibition versus [S] plot (from 6B). What type of inhibition do you observe?
 Graph double reciprocal plots (Lineweaver-Burk) of both of your experiments on the same
graph. Determine the actual and apparent Km and Vmax values. What type of inhibition do you
observe? Calculate the Ki.

Practice Problems (Not for lab report):

 Enzyme activity assays were performed with and without an inhibitor and the results are
graphed below. The values of the x and y intercepts are labeled.
o This is an example of what type of inhibition?
o Based on the graph, calculate Ki. Include units.

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WEEK 7
A. Generate Extracts Containing Lysozyme From Egg White
B. Perform a Bradford Assay to Determine Protein Concentration of the Egg Extract
C. Perform an Enzyme Activity Assay with M. luteus Cultures
D. Set Up Crystallization Trays With Purified Lysozyme

Definitions
Lysozyme:
A widely distributed enzyme that catalyzes the break down of specific polysaccharides,
including that found in bacterial cell walls.

Protein Crystallization:
The slow precipitation of a purified protein in solution into well-ordered crystals. This
process can occur by manipulating a number of variables in the protein solution,
including salt content, pH, temperature, and protein concentration.

Nucleation:
The process of forming a small microscopic cluster of proteins from which a crystal will
form. This requires supersaturation of the protein in solution.

Vapor Diffusion:
A method to crystallize a protein that involves a reservoir that contains a precipitant,
and a drop containing the protein of interest in a solution with lower levels of
precipitant. As time passes, water will leave the drop, increasing the protein and
precipitant concentration until the protein hopefully forms crystals.

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Lysozyme
Lysozyme is an example of a hydrolase, a class of enzymes that catalyze the hydrolysis
of chemical bonds. Specifically it is a glycosylase, an enzyme, which cleaves sugars.
Lysozyme is responsible for the cleavage of the beta(1-4) glycosidic bond between N-
acetylglucosamine (NAG) and N-acetylmuramic acid (NAM).

Figure 1. The lysozyme substrate. The beta (1-4) bonds circled are the targets for
hydrolysis. Taken from lysozyme.co.uk.

Alexander Fleming identified lysozyme in 1922, allegedly through nasal secretions that
fell onto a plate containing bacteria. The bacteria on the regions of the plate containing
the secretions dissolved, and through further investigation, lysozyme was identified as
the cause. We now know that lysozyme is present in a wide variety of prokaryotes and
eukaryotes, and that one of its targets are the NAG and NAM sugars which are major
components of the cell wall in bacteria and yeast. Hydrolysis of these sugars results in
weakening of the cell wall. Without a functional cell wall, osmotic stress causes the
plasma membranes of these cells to burst, leading to cell death.
Because of this function, lysozyme has a number of uses related to defense against
microbes. It is used as an antibacterial agent for food storage, a drug treatment for some
infections, and a food additive in certain milk products.
Lysozyme also has played a major role in biological research. Because it is such a highly
conserved enzyme and it is found in very high concentration in a variety of sources
(including chicken eggs and saliva), it has been an excellent subject for our
understanding of the relationship between protein structure and function. Lysozyme
was one of the first protein structures solved using X-ray diffraction, and because of the
ease of crystallization is still used by many labs today to troubleshoot crystallization
protocols.

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 *

Figure 2. Structure of Lysozyme. marks the enzymes active site where it binds to
the carbohydrate substrate and cleaves it into two. Taken from lysozyme.co.uk.

In today’s experiment, you will isolate proteins from a chicken egg, a major component
of which will be lysozyme. You will measure lysozyme activity using a sample of
Micrococcus luteus, a Gram positive bacterium, whose cell wall is susceptible to
breakdown by lysozyme. From this, you will determine the reaction rate and specific
activity of the egg white extract. This will also require performing a Bradford assay.
Finally, you will undertake a study in protein crystallization using lysozyme as the
subject. For the next few weeks you will act like a true crystallographer, altering a
number of variables to obtain the best crystals possible.

EXPERIMENT 7A – Generate extracts containing lysozyme

from egg whites

REAGENTS:
Egg
Cheesecloth
NAT Buffer (0.2M NaCl, 50mM Tris pH 9.0, 0.07% -mercaptoethanol)
Ice bucket
Beakers

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PROCEDURE:
 Crack the egg in half over a 50ml beaker. Isolate the egg white in the beaker by passing the
egg yolk from one half of the shell to the next. It is not necessary to get all of the egg white,
just as much as possible without contaminating it with the yolk.
 Pour the egg white into a double layer of cheesecloth, which is sitting in a 100ml beaker.
Close the cheesecloth around the egg white like a sack, and gently put pressure on the
cheesecloth so the egg white is filtered into the beaker. Collect as much as possible without
putting excessive force on the cheesecloth.
 Dispose what remains of the egg white and cheesecloth into the designated waste container.
 Remove 10ml of the egg white filtrate and add it into a 50ml conical tube. Label as egg white
extract along with your group information. If you have less than 10ml, record the amount
added.
 Combine with 15ml cold NAT buffer and gently rock the tube to mix the contents. If there is
less than 10ml egg white, add a proportionate amount less of the NAT buffer.
 Place the tube on ice for 30 minutes. Gently rock the tube every 5 minutes.

EXPERIMENT 7B – Bradford assay to determine protein

concentration
Next, you will perform a Bradford assay on the egg white extract to determine the
protein concentration. The procedure will be identical to the one you performed on your
P450 bacterial extracts.

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PROCEDURE:
Take a tube of bovine serum albumin (BSA) of concentration 0.5 mg/ml. For the assay
you want to have a final volume of 1.0ml for each sample, although since the amount of
BSA added will be very small, it does not need to be exact. Make your dilutions with
distilled water. Fill in the protocol charts below.

STANDARD CURVE USING BSA

Cuvette g BSA l BSA ml H2O ml Bradford Absorbance

reagent

Blank 0 0 0.8 0.2

#1 0.5 1 0.8 0.2
#2 1 0.8 0.2
#3 2 0.8 0.2
#4 4 0.8 0.2
#5 8 0.8 0.2

For your egg white extract, you will need to make a 1:10 dilution in water.

EGG WHITE EXTRACT

l cell free
ml Bradford
Cuvette extract (1:10 ml H2O Absorbance
reagent
dilution)
# 6 Egg White
1 0.8 0.2
Extract

Graph the BSA standards and determine the best-fit line. Use this to calculate the
concentration of your egg white extract.

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EXPERIMENT 7C – Perform an enzyme activity assay with
M. luteus cultures

As lysozyme is capable of hydrolyzing cell wall sugars, a measure of the enzyme activity
is based on its ability to lyse bacterial cells. Bacteria in a culture have a given optical
density corresponding to the amount of growth. More growth in a culture means that
there are more cells, and these cells will scatter light in the spectrophotometer,
producing a higher absorbance value. As lysozyme breaks down the cell walls of these
cells and lysis occurs, there will be fewer cells present and the optical density in the
culture will decrease. Thus, you can measure the activity of your lysozyme based on
how quickly the absorbance of a bacterial culture decreases over time. When working
with the M. luteus cells, you will have to dilute the original sample. Once diluted to the
appropriate density, work as quickly possible, since the M. luteus cells have a doubling
time of approximately 30 minutes. Waiting too long between performing your dilution
and the beginning of your experiment can throw off the starting optical density.

REAGENTS:
M. luteus culture
Egg white extract
0.1M potassium phosphate buffer pH 7

PROCEDURE:
 Obtain an M. luteus culture and determine its OD at 450nm in the spectrophotometer. To do
so, make a 1:10 dilution of the culture in LB (1ml total). Use 1ml LB as a blank.
 Based on the OD of your culture (be sure to factor in the 1:10 dilution), make 10ml of a 0.5 OD
cell solution in phosphate buffer. This will be used in your enzyme assay. Your initial
cell suspension will be on ice. Once you make your dilution, keep it at room temperature
for about 5 minutes before running your assay. If the reaction is still cold, the lysozyme
may not function at maximum efficiency.
 You will set up 2 tubes:
o Blank – 2ml phosphate buffer
o Egg white extract – 0.95ml of your 0.5OD cells + 50l of your egg white extract
o Note: Lysozyme is to be added last, immediately before beginning the assay. Like with
NADPH, once you add the lysozyme into in the cuvette, mix a few times, then place it
in the spectrophotometer.

 To run this assay, we can use the same program as the P450 enzyme activity assay. Open
this program, but change the wavelength from 340nm to 450nm.

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  Blank the spectrophotometer and add the lysozyme cuvettes into the spectrophotometer. For
a 2 minute interval, measure the absorbance at 450nm every 10 seconds.
 Look over your data. You would like to see roughly a 0.20 OD drop over the reaction. If it is
drastically more or less than this, you may need to repeat.

Based on experiments 7A-7C, you should be able to answer the following questions.
This will not be necessary for a lab report, but similar ideas will more than likely be
found on your final exam.
 What is the protein concentration of your egg white extract?
 Graph your data on an OD v. time plot. What is the reaction rate in terms of OD?
 For lysozyme, one enzyme unit (U) is defined as the amount of enzyme that causes an
absorbance decrease of 0.001 every minute (at pH 7 and 25C). For example, if you saw a
decrease of 0.10 per minute, there were 100U of activity in the 100l of extract that you added
to the cell suspension.
 Based on this, how many units of enzyme activity per ml are present in your enzyme extract?
 The specific activity of your egg white extract is defined as:

Units of lysozyme/mL
mg. of total protein/mL
What is the specific activity of your egg white extract? What units are attached to this value?

Protein Crystallography
Knowing the structure of a protein can tell us much about it. With the structure, we have
a greater understanding of the enzymatic activity being performed and the mechanism
by which it accomplishes it. If the protein is involved in a disease, knowing the structure
allows us to identify molecules that may be capable of interacting with and inhibiting
the protein. The structure can also help us to understand mutations, as we may be able
to visualize how the protein structure changes due to the modification.
There are many different ways to gain knowledge of a protein’s structure, including
mutagenesis to determine the importance of specific amino acids, homology to compare
a protein of interest to similar proteins in other organisms, and computer based tools to
model the structure of a protein based on its amino acid sequence. Two direct methods
to uncover a protein’s structure are X-ray crystallography and NMR (nuclear magnetic
resonance). NMR is a less invasive procedure, and can be performed with proteins in
solution. But the resolution of the structure obtained from NMR is much lower than that
with X-ray crystallography, and proteins also must be smaller than 30kD. X-ray
crystallography does not have these limitations, but for this process, protein crystals
must be obtained.

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So how are proteins crystallized? Crystallization is very much an art, as there is no
definitive method to obtain a crystal for a given protein. Crystallization varies with each
protein and may require solutions of different ion concentration or pH and incubation at
different temperatures. The crystallography process has been greatly aided by
molecular cloning procedures. The ability to express a foreign protein in other cells like
bacteria and yeast means it is possible to gain large quantities of your protein of interest.
Today, robotics is often used to assist in the crystallization process, as machines are
capable of testing numerous crystallization variables.
When crystallizing a protein, it is important that your starting material is pure. If not,
you may be crystallizing other proteins than the one you are interested in. For your
experiments, you will be working with pure lysozyme purchased from a company as
opposed to the lysozyme from your egg extract.
Protein crystals are formed in solution when the protein is supersaturated. There is a
fine line between a protein that is not saturated, where all the protein is in solution and
no crystals are formed, and one that is too saturated, where large amounts of the protein
will precipitate and you will see a large aggregate of protein as opposed to nicely formed
crystals.
Protein crystallization occurs when the protein is placed in a buffer with a compound
that will assist in crystal formation, known as a precipitant. As time passes, water
evaporates resulting in increased concentration of protein and precipitant. At some
point, the protein is supersaturated and crystals form. The steps involved are illustrated
by the phase diagram below:

Figure 3. Phase diagram detailing the steps and conditions

involved in protein crystallization. Taken from Olieric et al.
Biochemistry and Molecular Biology Education, 2007.

1. The protein is set up in crystallization trays under conditions of low protein and
precipitant concentration. At this point, no crystals form.
2. As time passes, the protein and precipitant concentration increases, resulting in a
step known as nucleation. Nucleation is the process of forming small microscopic
clusters of protein, which will seed the larger crystals that we will observe.

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 Nucleation requires high levels of supersaturation to overcome the energy barrier
involved with microcluster formation. If the supersaturation is too low, nucleation
will not happen in a reasonable amount of time (days or weeks). If supersaturation is
too high, proteins may precipitate and form disordered aggregates rather than
crystals. This region is known as the precipitation zone.
3. Assuming the crystallization conditions are relatively stable following nucleation, the
solution will enter the metastable zone and large, well-ordered protein crystals will
form from the microclusters.

The exact conditions for these different zones highlighted in the phase diagram are not
easy to quantify. But the behavior of crystals obtained under different conditions will
illustrate which zone your crystallization conditions are in.
To crystallize lysozyme, we will be
utilizing sitting drop vapor diffusion.
Sitting drop vapor diffusion involves a
reservoir that is filled with a large
volume of a solvent, known as the
precipitant. In the column above, a drop
containing a 1:1 mix of protein and
precipitant is added, and the top of the
set-up is sealed with tape. Initially, the
protein is not saturated in the solution.
Since the reservoir contains a higher concentration of precipitant, water will diffuse
from the drop to the reservoir, increasing the concentration of protein and precipitant in
the drop. At some point, crystals will hopefully form.
Each group will initially be given an aliquot of lysozyme and assigned a specific salt
(precipitant) to create the reservoir solution. Different concentrations of lysozyme will
be added to determine the effect of protein concentration on crystal formation. Next
week, you will observe whether crystals formed and share your information with the
other groups. Using each group’s data, you will decide which variables to adjust,
including precipitant used, pH, salt concentration and temperature.

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EXPERIMENT 7D – Set up crystallization trays with purified
lysozyme

REAGENTS:
Lysozyme stock solution (100mg/ml) in 0.05M NaOAc
Precipitant – NaCl, NaBr, NaNO3, (NH4)2SO4, NaOAc
1M Sodium acetate (NaOAc) pH 4.7
Sitting drop crystallization tray

PROCEDURE:
 Each group will be assigned a precipitant. Make 10ml of reservoir solution in a 15ml conical
tube. The reservoir solution will be 10% of your salt and 0.05M sodium acetate pH 4.7. Label
your reservoir solution with the contents and your group ID.
 You will use 4 different concentrations of lysozyme: 80mg/ml, 40mg/ml, 20mg/ml and
10mg/ml.
Starting with your stock solution, make 50l of 80mg/ml lysozyme in 0.05M sodium acetate.
Then perform serial dilutions to make the remaining 3 lysozyme solutions. Take 25l of the
80mg/ml solution and add 25l of sodium acetate to make the 40mg/ml solution. Use the
40mg/ml solution to make the 20mg/ml and so on.
 You also want to examine whether your egg white extract can be used for protein
crystallization. Based on the Bradford assay, determine the concentration of your extract. If
this concentration is greater than 80mg/ml, dilute it to 80mg/ml and create the same 4
concentrations as used with the pure lysozyme sample. If this concentration is less than
80mg/ml, use the extract as the highest concentration and perform two-fold serial dilutions
to generate the 4 different concentrations.

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  Set up your crystallization trays. Fill out the diagram below, labeling what is found in each
well.

 Add 750l of your reservoir solution to the reservoir of the crystallization tray.
 Add 7.5l of the 4 different lysozyme solutions to the column.
 Add 7.5l of reservoir solution to each drop in the colum. Do not pipette up and down to mix.
The two solutions will mix by diffusion. You can also gently stir the solution with your pipette
tip. Try to avoid introducing bubbles.
 Add 1.5l of Izit Protein Dye. There is no need to mix, as the dye will diffuse on its own.
 Place tape over the wells containing protein. Close the lid on the tray and wait until next week
to check for crystals.

Practice Problem
A precipitant solution is to consist of 20% NaBr, 5% DMSO and 25mM NaOAc. If a student
wants to make 50ml of this solution, and she has solid NaBr, a 75% DMSO solution and a
2M NaOAc solution, how will she create her precipitant solution?

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WEEK 8
A. Observe Crystal Formation
B. Devise New Conditions to Crystallize Lysozyme

Definitions
Izit Protein Dye:
Izit is a small molecular dye which allows you to distinguish between protein crystals
and crystals of inorganic matter, such as salts. Protein crystals consist largely of water
(40-60%) and thus contain large solvent channels that the Izit dye can penetrate. Thus,
protein crystals will appear a dark blue when they take up the dye whereas salt crystals
will look no darker than the surrounding fluid.

Last week you set up wells in hopes of obtaining lysozyme crystals. Looking under the
4x magnification on your microscope, make notes of what you observe, and then
compare your work with that of the other groups in the class. With this information,
determine what precipitant(s) work best for crystallization. This week you will expand
the possible variables to test, including pH and temperature.
Based on the choices you make, you will write an introduction for a protein
crystallization lab report. An entire lab report will not be written, just the introduction to
be turned in by lab next week.

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Microscope basics

Reference: In Models CHS/CHT Biological Microscope Instructions . Edited by Olympus Optical

Co., LTD.. pp. 3-4. , Printed in Japan 9204 M 30. Tokyo, Japan.

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The Revolving Nosepiece rotates through the four Objectives: 4x, 10x, 40x, and 100x (oil
immersion lens) objectives. The 4x objective will be sufficient for our experiment.
The Coarse Adjustment Knob is used to bring the specimen into view only with the 10x
objective lens. It should not be used with the 40x and 100x objectives.
The Condenser contains lenses that focus light maximally onto the specimen.
A Voltage Control Dial controls the intensity of light that is being emitted by the light
source.
The following procedure is how to focus your crystals with the microscope:
1. Place the tray on the stage of the microscope. Due to the size of the tray you may need
to adjust its position to view each individual well. Turn on the illumination and
position the well of interest over the slot in the stage.
2. Find your crystals with 4x objective and bring it into focus using the course
adjustment. Then use the fine adjustment knob to get a sharper image.

Using the Ocular Micrometer in your Microscope

It is important to get an idea of the size of your crystals. This is accomplished by using
the ocular micrometer which has been inserted into the right ocular (eyepiece) of your
microscope. The physical length of the ocular unit depends on which objective lens is
being used, that is, on the degree of magnification. The ocular micrometer is stationary;
therefore, you must move the object to the micrometer in order to make a measurement.

To determine the length that each ocular unit represents, divide 100 by the power of
each objective. For example, to calculate the value of each unit when using the 10X
objective, divide 100 by 10 to obtain the answer 10 microns.

Example:

Here we depict a molecule measuring 50 microns

in length, viewed at three different
magnifications:
Objective Lens Used
10X - each ocular unit is equivalent to 10
microns
4X - each ocular unit is equivalent to 25 microns

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EXPERIMENT 8A – Observe crystal formation

PROCEDURE:
 Observe each well that you set up the previous week. What difference did you see in the
wells with the pure lysozyme versus the egg white extract?
 Focus your observations on the wells with pure lysozyme. On the diagram below, describe
what you observe, including an approximate size of the crystals using the ocular micrometer.
Draw an example of a crystal. Notice the color of the crystals. Protein crystals will be darker,
as they have taken up the Izit dye. Salt crystals will be the same color as the rest of the
solution.

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 Pass your tray to the next group. You will observe the results from each other group with the
precipitant they used. Take notes on these results.

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Crystallization Variables
The main goal in crystallization is to create an environment, which will result in
supersaturation of your protein. Too little supersaturation and crystal nucleation will
not occur. Too much supersaturation can cause excess nucleation to occur resulting in
many small crystals or protein aggregation resulting in disorganized protein molecules
rather than well-ordered crystals.
A number of variables can alter crystal formation, which will be discussed below.
pH
Protein solubility is minimal at the protein’s isoelectric point (pI). The pI is the pH at
which a given protein has no net electrical charge. At the protein’s pI, it is not subject to
salting in, as the salt is more likely to interact with water instead of the protein. As you
move the pH away from the protein’s pI, solubility increases in solutions of low salt.
This is due to excess H+ or OH- ions, which steal or donate H+ to the protein’s amino
acids. The increased charges on the protein are capable of interacting with water
molecules that surround and solubilize it. At extreme pH’s though, the charges on the
protein may increase too much, and the proteins are more likely to interact with each
other, aggregate and precipitate out of solution. These characteristics can be exploited to
assist in protein crystallization.

Type of Salt and Concentration

Crystallization depends on being able to salt out the protein. But the salting out
properties of a protein vary based on the protein’s amino acid composition and the salt
being used. Ammonium sulfate is a commonly used salt as it has a high solubility in
water, which means you can generate a solution with a very high ionic strength.
Another consideration is the charge of the molecule, as a divalent ion will produce a
higher ionic strength compared to a molecule with only a single charge. Certain ions are
capable of decreasing the solubility of a protein and stabilizing their conformation in the
process to ensure they are not denatured. Other ions actually increase the solubility of a
protein.

Temperature
Crystallization is usually performed at room temperature or at 4C. Higher
temperatures, generally over 40C, tend to denature a protein although proteins such as
glucagon have been crystallized as high as 60C. Higher temperatures also may be
beneficial when dealing with proteins from thermophilic organisms. Temperature
affects crystallization because it affects protein solubility. So it is important to take into
consideration the protein’s solubility at a given temperature before considering how

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that environment may affect crystallization. Another thing to remember is that
molecules diffuse more slowly at low temperatures, so the vapor diffusion process and
thus crystal formation may take longer when incubated in the cold. In your
experiments, feel free to test out crystallization at 4C, room temperature or 37C.

EXPERIMENT 8B – Devise new conditions to crystallize

lysozyme
Based on the class results, you should be looking for a number of characteristics,
including:
1. In each condition, did protein crystals form? Salt crystals?
2. Using the micrometer, what was the approximate size of an average crystal in each
well?
3. About how many crystals were formed in each well?
With this information, your group will now devise a new strategy to crystallize the
lysozyme protein. Your goal should be to obtain a small number of fairly large, well
ordered protein crystals. Higher quality crystals contain few facets that are smooth. So a
cube shaped crystal with clean sides is preferable to a crystal that has 16 sides and
cracks throughout. If you already achieved a high quality crystal in some of your wells,
tweak your conditions to test the effect of changes in pH or temperature on the
crystallization process. Or start with a less “polished” precipitant to attempt to improve
the crystals by altering the conditions. Also, keep in mind that experimental error can
play a role in crystal formation. This is not to say that you should distrust your or your
classmates’ results, but that the results obtained are not completely definitive. Repeating
a given set of conditions will not guarantee the exact same outcome.
In total, you will set-up 8 different crystallization conditions, each with varying
concentrations of lysozyme (80mg/ml and 40mg/ml). This means in total you will be
setting up 16 wells. You may start with any salt as precipitant, it is not necessary to test
the same one as last week. Keep in mind the goal is to determine how variables effect
crystallization. So choosing “perfect” crystal conditions as your starting point may mean
all of your crystals will turn out similarly regardless of changing conditions.
Below are the potential variables you can change:
 Precipitant salt – Any of the 5 salts tested in Week 7
 Precipitant concentration – Ranging from 2% to 20% salt in 0.05M sodium acetate
 pH of precipitant solution – 1M Sodium Acetate stocks with pH 4.0, 4.6, or 5.0
 Crystallization temperature – Incubate trays at 4C, room temperature, or 37C
Keep in mind that in all controlled experiments, only 1 variable is altered at a time.

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For example, if you wanted to test crystallization temperature, you would set up two
trays with the same precipitant solution (same salt, salt concentration and pH), but one
would be placed at room temperature and one at 37C.
If you wanted to test how a different salt and different temperature affected
crystallization, you would need 4 conditions:
(1) standard precipitant//temperature
(2) different salt/same temperature as used in (1)
(3) same salt/different temperature as used in (1)
(4) different salt (the one used in (2))/different temperature (the one used in (3))
In this way, it is possible to conclude that a specific variation caused a change in crystal
formation. The tables below will help you to set up your crystallization conditions for
this week.
For each condition, make 2ml precipitant. If you plan on using the same precipitant in
multiple trays (for example, when only temperature is a variable), scale up the volume
of precipitant made accordingly.
Precipitant and protein solution volumes are to be the same as last week:
 750l precipitant in the reservoir
 7.5l lysozyme stock (either 80 or 40mg/ml) in the well
 7.5l precipitant in the well
 1.5l Izit protein dye

Use the tables below to map out your crystallization strategy. Discuss your plan with
your TA before making solutions.
After setting up each condition, tape the tops of the wells used and cover the tray with
the lid. Place at the appropriate temperature until next week.
Condition 1 - Control

Salt Salt concentration 1M NaOAC pH Temperature

Recipe for precipitant solution:

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Condition 2 – Compare to condition(s) #

Salt Salt concentration 1M NaOAC pH Temperature

Recipe for precipitant solution:

Condition 3– Compare to condition(s) #

Salt Salt concentration 1M NaOAC pH Temperature

Recipe for precipitant solution:

Condition 4 – Compare to condition(s) #

Salt Salt concentration 1M NaOAC pH Temperature

Recipe for precipitant solution:

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Condition 5 – Compare to condition(s) #

Salt Salt concentration 1M NaOAC pH Temperature

Recipe for precipitant solution:

Condition 6 – Compare to condition(s) #

Salt Salt concentration 1M NaOAC pH Temperature

Recipe for precipitant solution:

Condition 7 – Compare to condition(s) #

Salt Salt concentration 1M NaOAC pH Temperature

Recipe for precipitant solution:

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Condition 8 – Compare to condition(s) #

Salt Salt concentration 1M NaOAC pH Temperature

Recipe for precipitant solution:

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WEEK 9
Observe Crystal Formation and Fill Out the Crystallization Worksheet

Observe each well that you set up the previous week. Each group will be given a
worksheet to fill out regarding your results which to be completed by the end of this
class period. All group members are to participate in filling out the worksheet to earn
full credit.

EXPERIMENT 9 – Observe crystal formation and fill out the

Crystallization worksheet

Again, observe the crystallization trays your group set up. You should be looking for a
number of characteristics including:
1. In each condition, did protein crystals form? Salt crystals?
2. Using the micrometer, what was the approximate size of an average crystal in each
well?
3. About how many crystals were formed in each well?

Based on your results, fill out the crystallization worksheet provided by your TA. This
will be filled out today and handed in (1 per group) before the end of the lab period.

Congratulations on completing Biochemistry lab! Prepare for the final (stop by office
hours or email any questions you have), stay calm while taking it, and good luck on your
other classes.

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